Targeting Trypanothione Metabolism in Trypanosomatids.

Infectious diseases caused by trypanosomatids, including African trypanosomiasis (sleeping sickness), Chagas disease, and different forms of leishmaniasis, are Neglected Tropical Diseases affecting millions of people worldwide, mainly in vulnerable territories of tropical and subtropical areas. In general, current treatments against these diseases are old-fashioned, showing adverse effects and loss of efficacy due to misuse or overuse, thus leading to the emergence of resistance. For these reasons, searching for new antitrypanosomatid drugs has become an urgent necessity, and different metabolic pathways have been studied as potential drug targets against these parasites. Considering that trypanosomatids possess a unique redox pathway based on the trypanothione molecule absent in the mammalian host, the key enzymes involved in trypanothione metabolism, trypanothione reductase and trypanothione synthetase, have been studied in detail as druggable targets. In this review, we summarize some of the recent findings on the molecules inhibiting these two essential enzymes for Trypanosoma and Leishmania viability.

Miltefosine and Nifuratel Combination: A Promising Therapy for the Treatment of Leishmania donovani Visceral Leishmaniasis.

Visceral leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania donovani and Leishmania infantum that is endemic not only in East African countries, but also in Asia, regions of South America and the Mediterranean Basin. For the pharmacological control of this disease, there is a limited number of old and, in general, poorly adherent drugs, with a multitude of adverse effects and low oral bioavailability, which favor the emergence of resistant pathogens. Pentavalent antimonials are the first-line drugs, but due to their misuse, resistant Leishmania strains have emerged worldwide. Although these drugs have saved many lives, it is recommended to reduce their use as much as possible and replace them with novel and more friendly drugs. From a commercial collection of anti-infective drugs, we have recently identified nifuratel-a nitrofurantoin used against vaginal infections-as a promising repurposing drug against a mouse model of visceral leishmaniasis. In the present work, we have tested combinations of miltefosine-the only oral drug currently used against leishmaniasis-with nifuratel in different proportions, both in axenic amastigotes from bone marrow and in intracellular amastigotes from infected Balb/c mouse spleen macrophages, finding a potent synergy in both cases. In vivo evaluation of oral miltefosine/nifuratel combinations using a bioimaging platform has revealed the potential of these combinations for the treatment of this disease.

Antileishmanial Effect of 1,5- and 1,8-Substituted Fused Naphthyridines.

In the absence of a vaccine, there is a need to find new drugs for the treatment of neglected tropical diseases, such as leishmaniasis, that can overcome the many drawbacks of those currently used. These disadvantages include cost, the need to maintain a cold chain, the route of administration, the associated adverse effects and the generation of resistance. In this work we have evaluated the antileishmanial effect of 1,5- and 1,8-substituted fused naphthyridines through in vitro and ex vivo assays, using genetically modified axenic and intramacrophagic Leishmania infantum amastigotes. The toxicity of these compounds has been tested in the mammalian host cell using murine splenic macrophages, as well as in murine intestinal organoids (miniguts) in order to assess their potential for oral administration. The 1,8- derivatives showed greater leishmanicidal activity and the presence of a nitrogen atom in the fused ring to the naphthyridine was important to increase the activity of both types of molecules. The aromatization of the pyridine ring also had marked differences in the activity of the compounds.

DNA flowerstructure co-localizes with human pathogens in infected macrophages.

Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology.

Novel phenanthridine amide analogs as potential anti-leishmanial agents: In vitro and in silico insights.

In the current work, sixteen novel amide derivatives of phenanthridine were designed and synthesized using 9-fluorenone, 4-Methoxy benzyl amine, and alkyl/aryl acids. The characterization of the title compounds was performed using LCMS, elemental analysis, 1HNMR, 13CNMR and single crystal XRD pattern was also developed for compounds A8. All the final analogs were screened in vitro for anti-leishmanial activity against promastigote form of L. infantum strain. Among the tested analogs, four compounds (A-06, A-11, A-12, and A-15) exhibited significant anti-leishmanial activity with EC50 value ranges from 8.9 to 21.96 µM against amastigote forms of tested L. infantum strain with SI ranges of 1.0 to 4.3. From the activity results it was found that A-11 was the most active compound in both promastigote and amastigotes forms with EC50 values 8.53 and 8.90 µM respectively. In-silico ADME prediction studies depicted that the titled compounds obeyed Lipinski's rule of five as that of the approved marketed drugs. The predicted in-silico toxicity profile also confirmed that the tested compounds were non-toxic. Finally, molecular docking and molecular dynamics study was also performed for significantly active compound (A-11) in order to study it's putative binding pattern at the active site of the selected leishmanial trypanothione reductase target as well as to understand the stability pattern of target-ligand complex for 100 ns. Single crystal XRD of compound A-08 revealed that the compound crystallizes in monoclinic C2/c space group and showed interesting packing arrangements.

Drug discovery technologies: Caenorhabditis elegans as a model for anthelmintic therapeutics.

Helminthiasis is one of the gravest problems worldwide. There is a growing concern on less available anthelmintics and the emergence of resistance creating a major threat to human and livestock health resources. Novel and broad-spectrum anthelmintics are urgently needed. The free-living nematode Caenorhabditis elegans could address this issue through automated high-throughput technologies for the screening of large chemical libraries. This review discusses the strong advantages and limitations for using C elegans as a screening method for anthelmintic drug discovery. C elegans is the best model available for the validation of novel effective drugs in treating most, if not all, helminth infections, and for the elucidation the mode of action of anthelmintic candidates. This review also focuses on available technologies in the discovery of anthelmintics published over the last 15 years with particular attention to high-throughput technologies over conventional screens. On the other hand, this review highlights how combinatorial and nanomedicine strategies could prolong the use of anthelmintics and control resistance problems.

Druggable targets of SARS-CoV-2 and treatment opportunities for COVID-19.

COVID-19 caused by the novel SARS-CoV-2 has been declared a pandemic by the WHO is causing havoc across the entire world. As of May end, about 6 million people have been affected, and 367 166 have died from COVID-19. Recent studies suggest that the SARS-CoV-2 genome shares about 80% similarity with the SARS-CoV-1 while their protein RNA dependent RNA polymerase (RdRp) shares 96% sequence similarity. Remdesivir, an RdRp inhibitor, exhibited potent activity against SARS-CoV-2 in vitro. 3-Chymotrypsin like protease (also known as Mpro) and papain-like protease, have emerged as the potential therapeutic targets for drug discovery against coronaviruses owing to their crucial role in viral entry and host-cell invasion. Crystal structures of therapeutically important SARS-CoV-2 target proteins, namely, RdRp, Mpro, endoribonuclease Nsp15/NendoU and receptor binding domain of CoV-2 spike protein has been resolved, which have facilitated the structure-based design and discovery of new inhibitors. Furthermore, studies have indicated that the spike proteins of SARS-CoV-2 use the Angiotensin Converting Enzyme-2 (ACE-2) receptor for its attachment similar to SARS-CoV-1, which is followed by priming of spike protein by Transmembrane protease serine 2 (TMPRSS2) which can be targeted by a proven inhibitor of TMPRSS2, camostat. The current treatment strategy includes repurposing of existing drugs that were found to be effective against other RNA viruses like SARS, MERS, and Ebola. This review presents a critical analysis of druggable targets of SARS CoV-2, new drug discovery, development, and treatment opportunities for COVID-19.

Screening Marine Natural Products for New Drug Leads against Trypanosomatids and Malaria.

Neglected Tropical Diseases (NTD) represent a serious threat to humans, especially for those living in poor or developing countries. Almost one-sixth of the world population is at risk of suffering from these diseases and many thousands die because of NTDs, to which we should add the sanitary, labor and social issues that hinder the economic development of these countries. Protozoan-borne diseases are responsible for more than one million deaths every year. Visceral leishmaniasis, Chagas disease or sleeping sickness are among the most lethal NTDs. Despite not being considered an NTD by the World Health Organization (WHO), malaria must be added to this sinister group. Malaria, caused by the apicomplexan parasite Plasmodium falciparum, is responsible for thousands of deaths each year. The treatment of this disease has been losing effectiveness year after year. Many of the medicines currently in use are obsolete due to their gradual loss of efficacy, their intrinsic toxicity and the emergence of drug resistance or a lack of adherence to treatment. Therefore, there is an urgent and global need for new drugs. Despite this, the scant interest shown by most of the stakeholders involved in the pharmaceutical industry makes our present therapeutic arsenal scarce, and until recently, the search for new drugs has not been seriously addressed. The sources of new drugs for these and other pathologies include natural products, synthetic molecules or repurposing drugs. The most frequent sources of natural products are microorganisms, e.g., bacteria, fungi, yeasts, algae and plants, which are able to synthesize many drugs that are currently in use (e.g. antimicrobials, antitumor, immunosuppressants, etc.). The marine environment is another well-established source of bioactive natural products, with recent applications against parasites, bacteria and other pathogens which affect humans and animals. Drug discovery techniques have rapidly advanced since the beginning of the millennium. The combination of novel techniques that include the genetic modification of pathogens, bioimaging and robotics has given rise to the standardization of High-Performance Screening platforms in the discovery of drugs. These advancements have accelerated the discovery of new chemical entities with antiparasitic effects. This review presents critical updates regarding the use of High-Throughput Screening (HTS) in the discovery of drugs for NTDs transmitted by protozoa, including malaria, and its application in the discovery of new drugs of marine origin.

Antiparasitic effect of synthetic aromathecins on Leishmania infantum.

BACKGROUND: Canine leishmaniasis is a zoonotic disease caused by Leishmania infantum, being the dogs one of the major reservoirs of human visceral leishmaniasis. DNA topology is a consolidated target for drug discovery. In this regard, topoisomerase IB - one of the enzymes controlling DNA topology - has been poisoned by hundreds of compounds that increase DNA fragility and cell death. Aromathecins are novel molecules with a multiheterocyclic ring scaffold that have higher stability than camptothecins. RESULTS: Aromathecins showed strong activity against both forms of L. infantum parasites, free-living promastigotes and intra-macrophagic amastigotes harbored in ex vivo splenic explant cultures obtained from infected BALB/c mice. However, they prevented the relaxation activity of leishmanial topoisomerase IB weakly, which suggests that the inhibition of topoisomerase IB partially explains the antileishmanial effect of these compounds. The effect of aromathecins was also studied against a strain resistant to camptothecin, and results suggested that the trafficking of these compounds is not through the ABCG6 transporter. CONCLUSIONS: Aromathecins are promising novel compounds against canine leishmaniasis that can circumvent potential resistances based on drug efflux pumps.

Plant-based larvicidal agents: An overview from 2000 to 2018.

Current review aims to systematically segregate, analyze and arrange the key findings of the scientific reports published on larvicidal plants including larvicidal formulations. The investigation was carried out by analyzing the published literature in various scientific databases, subsequently, the key findings of the selective scientific reports having larvicidal potency (LC50) of extract or isolated oil<100 µg/mL were tabulated to provide the concise and crucial information. Special emphasis was given on reports in which LC50 of extract or isolated oil was reported to be < 10 µg/mL, genus or species documented in multiple independent studies, advancement in larvicidal formulations and activity of isolated phytoconstituents. Extensive analysis of published literature revealed that the larvicidal potency of herbal resources varied from sub-microgram/ml to practically insignificant. Overall, this unprecedented summarized and arranged information can be utilized for design, development and optimization of herbal based formulation having potential larvicidal activity.

Synthesis and activity of benzopiperidine, benzopyridine and phenyl piperazine based compounds against Leishmania infantum.

In the present study, anti-leishmanial evaluation of twenty four structurally diverse compounds based on benzopiperidine, benzopyridine and phenylpiperazine nucleuses against Leishmania infantum has been reported. Cytotoxicity studies of all the compounds were performed on murine non-infected splenocytes. Tested compounds exhibited weak to potent activity against promastigote (IC50 3.21 ±â€¯1.40 to >100 µM) as well as amastigote (IC50 6.84 ±â€¯2.5 to 92.47 ±â€¯17.61 µM) forms of tested strains. Moreover, two compounds F13 and F15 exhibited potent activity (IC50 < 10 µM) against both forms of the parasite with selectivity index ranges from 11.40 to 22.10. Overall, the current study afforded few hits with novel anti-leishmanial activity in low micromolar concentration, further hit optimization studies can be performed to get more potent candidates against the selected species of parasite.

Promising effects of 1,8 Cineole to control Giardia lamblia infection: Targeting the inflammation, oxidative stress, and infectivity.

Reportedly, synthetic drugs such as metronidazole, furazolidone, tinidazole, and quinacrine are used for the treatment of giardiasis but are associated with adverse effects. In this study, we aimed to investigate the in vitro and in vivo effects of eucalyptol (ECT, 1,8 cineole) alone and in combination with metronidazole (MNZ) on Giardia lamblia. The effects of ECT on cell viability, plasma membrane permeability, and gene expression levels of adenylate cyclase (AK) and extracellular signal kinases 1 and 2 (ERK1 and ERK2) in trophozoites of G. lamblia were assessed. In vivo, the effects of ECT alone and in combination with MNZ were assessed on mice infected with G. lamblia. In addition, the gene expression of inflammatory genes (e.g., TNF-α, IL-1ß, and IL-10) and antioxidant genes (catalase (CAT), superoxide dismutase 1 (SOD1), glutathione peroxidase 2 (GPX2)) was determined by real-time PCR. The IC50 values of ECT, MNZ, and ECT+MNZ on trophozoites were 30.2 µg/mL, 21.6 µg/mL, and 8.5 µg/mL, respectively. The estimated Fractional inhibitory concentration index (FICI) values for ECT and MNZ were 0.28 and 0.39, respectively. The application of ECT on G. lamblia trophozoites resulted in a dose-dependent increase in plasma membrane permeability, particularly at concentrations of ½ IC50 and IC50 (P < 0.05). The treatment of infected mice with various doses of ECT, mainly in combination with MNZ for 7 days, resulted in a significant decrease (P < 0.001) in the average number and viability of cysts. ECT, especially when combined with MNZ, caused a significant (P < 0.001) reduction in the expression of TNF-α and IL-6 genes, and an increase (P < 0.05) in the expression of IL-10 genes. ECT alone and mainly in combination with MNZ leads to a significant (P < 0.001) increase in the gene expression of CAT, SOD, and GPX genes. These findings demonstrate that the use of ECT in these doses, even for 14 days, does not have any toxic effects on the function of vital liver and kidney tissues. The study findings confirmed the promising effects of ECT against G. lamblia infection both in vitro and in vivo. Considering the possible mechanisms, ECT increases plasma membrane permeability and reduces the expression levels of infectivity-related genes. In addition, ECT suppresses inflammation and oxidative stress, controlling giardiasis in mice. More studies are needed to clarify these findings.

Treatment and Healing of Leishmaniasis in a Wolf in Semi-Captivity Regime from an Educational Center of Zamora Province (Spain).

Leishmaniasis in wild canids is a vector-borne disease caused in Europe by the protozoan parasite Leishmania infantum. To date, there is limited information on clinical signs and laboratory abnormalities in wolves due to leishmaniasis. The current clinical case report described a female Iberian wolf (Canis lupus signatus) housed in semi-captivity conditions at the Centro del Lobo Ibérico "Félix Rodríguez de la Fuente", in Robledo de Sanabria, Zamora (Spain), with an interdigital ulcerous wound at the right forepaw, hyper-gammaglobulinemia, and abnormal liver blood parameters. Definitive serodiagnosis of leishmaniasis was established using antileishmanial serum antibodies and PCR analysis of different biological samples. A gold-standard anti-L. infantum treatment protocol consisting in subcutaneous meglumine antimoniate and oral allopurinol combination was installed. However, the presence of pain at the site of injection due to meglumine antimoniate administration forced its substitution by oral miltefosine. A progressive reduction of the levels of anti-L. infantum serum antibodies and the concentrations of gamma-globulin fraction was detected after antileishmanial treatment as well as a decline of liver GPT. To our knowledge, this is the first case of leishmaniasis diagnosed in a wolf housed in semi-captivity conditions, with the condition subsequently treated and successfully cured.

Polyamine Metabolism for Drug Intervention in Trypanosomatids.

Neglected tropical diseases transmitted by trypanosomatids include three major human scourges that globally affect the world's poorest people: African trypanosomiasis or sleeping sickness, American trypanosomiasis or Chagas disease and different types of leishmaniasis. Different metabolic pathways have been targeted to find antitrypanosomatid drugs, including polyamine metabolism. Since their discovery, the naturally occurring polyamines, putrescine, spermidine and spermine, have been considered important metabolites involved in cell growth. With a complex metabolism involving biosynthesis, catabolism and interconversion, the synthesis of putrescine and spermidine was targeted by thousands of compounds in an effort to produce cell growth blockade in tumor and infectious processes with limited success. However, the discovery of eflornithine (DFMO) as a curative drug against sleeping sickness encouraged researchers to develop new molecules against these diseases. Polyamine synthesis inhibitors have also provided insight into the peculiarities of this pathway between the host and the parasite, and also among different trypanosomatid species, thus allowing the search for new specific chemical entities aimed to treat these diseases and leading to the investigation of target-based scaffolds. The main molecular targets include the enzymes involved in polyamine biosynthesis (ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine synthase), enzymes participating in their uptake from the environment, and the enzymes involved in the redox balance of the parasite. In this review, we summarize the research behind polyamine-based treatments, the current trends, and the main challenges in this field.

In Vitro and Ex Vivo Synergistic Effect of Pyrvinium Pamoate Combined with Miltefosine and Paromomycin against Leishmania.

One of the major drawbacks of current treatments for neglected tropical diseases is the low safety of the drugs used and the emergence of resistance. Leishmaniasis is a group of neglected diseases caused by protozoa of the trypanosomatidae family that lacks preventive vaccines and whose pharmacological treatments are scarce and unsafe. Combination therapy is a strategy that could solve the above-mentioned problems, due to the participation of several mechanisms of action and the reduction in the amount of drug necessary to obtain the therapeutic effect. In addition, this approach also increases the odds of finding an effective drug following the repurposing strategy. From the previous screening of two collections of repositioning drugs, we found that pyrvinium pamoate had a potent leishmanicidal effect. For this reason, we decided to combine it separately with two clinically used leishmanicidal drugs, miltefosine and paromomycin. These combinations were tested in axenic amastigotes of Leishmania infantum obtained from bone marrow cells and in intramacrophagic amastigotes obtained from primary cultures of splenic cells, both cell types coming from experimentally infected mice. Some of the combinations showed synergistic behavior, especially in the case of the combination of pyrvinium pamoate with paromomycin, and exhibited low cytotoxicity and good tolerability on intestinal murine organoids, which reveal the potential of these combinations for the treatment of leishmaniasis.

New benzimidazole derivative compounds with in vitro fasciolicidal properties.

BACKGROUND: Control of the zoonotic food-borne parasite Fasciola hepatica remains a major challenge in humans and livestock. It is estimated that annual economic losses due to fasciolosis can reach US$3.2 billion in agriculture and livestock. Moreover, the wide distribution of drug-resistant parasite populations and the absence of a vaccine threaten sustainable control, reinforcing the need for novel flukicides. METHODS: The present work analyses the flukicidal activity of a total of 70 benzimidazole derivatives on different stages of F. hepatica. With the aim to select the most potent ones, and screenings were first performed on eggs at decreasing concentrations ranging from 50 to 5 µM and then on adult worms at 10 µM. Only the most effective compounds were also evaluated using a resistant isolate of the parasite. RESULTS: After the first screenings at 50 and 10 µM, four hit compounds (BZD31, BZD46, BZD56, and BZD59) were selected and progressed to the next assays. At 5 µM, all hit compounds showed ovicidal activities higher than 71% on the susceptible isolate, but only BZD31 remained considerably active (53%) when they were tested on an albendazol-resistant isolate, even with values superior to the reference drug, albendazole sulfoxide. On the other hand, BZD59 displayed a high motility inhibition when tested on adult worms from an albendazole-resistant isolate after 72 h of incubation. CONCLUSIONS: BZD31 and BZD59 compounds could be promising candidates for the development of fasciolicidal compounds or as starting point for the new synthesis of structure-related compounds.

A pentapeptide signature motif plays a pivotal role in Leishmania DNA topoisomerase IB activity and camptothecin sensitivity.

BACKGROUND: Leishmania donovani - the causative agent of visceral leishmaniasis - has several evolutionary characteristics that make the disease difficult to combat. Among these differences, a rare heterodimeric DNA topoisomerase IB has been reported thus opening a new promising field in the therapy of leishmaniasis. Several studies of the human enzyme have pointed to the importance of the linker domain in respect to camptothecin sensitivity. At present, it has been impossible to pinpoint the regions that make up the linker domain in Leishmania. METHODS: Several site-directed mutations as well as internal and linear truncations involving both subunits were assayed on both, relaxation activity and sensitivity to camptothecin. RESULTS: Truncations performed on the trypanosomatids conserved motif (RPPVVRS) of the small subunit of leishmanial DNA topoisomerase IB demonstrated that elimination of pentapeptide RPPVV produced a nonfunctional enzyme. However, the removal of the dipeptide RS led to an enzyme with reduced relaxation activity and less sensitivity to camptothecin. The basic structure, both sensitive to camptothecin and able to fully relax DNA, composed of amino acids 1-592 and 175-262 in the large and small subunits, respectively. CONCLUSION: It has been established that the region between amino acids 175 and 180 (RPPVV) of the small subunit plays a pivotal role in both interaction with the large subunit and sensitivity to camptothecin in Leishmania. GENERAL SIGNIFICANCE: The present report describes a functional analysis of the leishmanial DNA topoisomerase IB regions directly involved both in sensitivity to poisons and in the conformation of the linker domain.

Synthesis of marine α-methoxylated fatty acid analogs that effectively inhibit the topoisomerase IB from Leishmania donovani with a mechanism different from that of camptothecin.

Sponges biosynthesize α-methoxylated fatty acids with unusual biophysical and biological properties and in some cases they display enhanced anticancer activities. However, the antiprotozoal properties of the α-methoxylated fatty acids have been less studied. In this work, we describe the total synthesis of (5Z,9Z)-(±)-2-methoxy-5, 9-eicosadienoic acid (1) and its acetylenic analog (±)-2-methoxy-5,9-eicosadiynoic acid (2), and report that they inhibit (EC50 values between 31 and 22 µM) the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB). The inhibition of LdTopIB (EC50 = 53 µM) by the acid (±)-2-methoxy-6-icosynoic acid (12) was studied as well. The potency of LdTopIB inhibition followed the trend 2 > 1 > 12, indicating that the effectiveness of inhibition depends on the degree of unsaturation. All of the studied α-methoxylated fatty acids failed to inhibit the human topoisomerase IB enzyme (hTopIB) at 100 µM. However, the α-methoxylated fatty acids were capable of inhibiting an active but truncated LdTopIB with which camptothecin (CPT) cannot interact suggesting that the methoxylated fatty acids inhibit LdTopIB with a mechanism different from that of CPT. The diunsaturated fatty acids displayed low cytotoxicity towards Leishmania infantum promastigotes (EC50 values between 260 and 240 µM), but 12 displayed a better cytotoxicity towards Leishmania donovani promastigotes (EC50 = 100 µM) and a better therapeutic index.

Secretory IgA as Biomarker for Gastrointestinal Nematodes Natural Infection in Different Breed Sheep.

Specific IgA antibody has been shown to play an important role in resistance to gastrointestinal nematode (GIN) infections in sheep, particularly in Teladorsagia circumcincta parasitosis. In some breeds, negative associations have been shown between IgA levels and worm burden in experimentally infected sheep. In the present study, we have studied the relationship between IgA levels in naturally infected sheep (582 ewes in total; 193 younger than one year old and 389 older than one year old) and fecal egg count (FEC) in the Assaf, Castellana, and Churra breeds. ELISA assays were performed to measure IgA levels against the somatic antigen of T. circumcincta third larval stage (L3) and a 203-amino-acid fragment of the protein disulfide isomerase from the same GIN species. A multilevel random intercept model was developed to predict the infection risk according to age or breed. Spearman's correlation rank was used for statistical analysis. The prediction model showed that breed was not an influential factor in this study, although the Assaf breed could be considered slightly more susceptible than the others. In addition, age affected the infection risk, with the young ewes more susceptible to infection than the adult groups, except for the Castellana breed, whose risk of infection was similar at all ages. The most significant positive association was found between FEC and IgA measured in the nasal secretions of young ewes using both antigens (Rho = 0.5; p = 0.00); the correlation of FEC with IgA in serum was moderately significant (Rho = 0.306; p = 0.00). Comparing both antigens, the protein disulfide isomerase antigen was less reactive than the somatic antigen from L3. In conclusion, under natural conditions, specific IgA against GIN was positively associated with FEC in sheep, with nasal secretions from young animals being the sample where this association is stronger, which, therefore, could be used as a marker of infection in further studies.

Inspection of in-house designed novel thiochromone amino-acid conjugate derivatives as Lm-NMT inhibitor - An in-silico analysis.

Leishmaniasis is a complex neglected tropical disease caused by various leishmanial parasites that primarily affect the world's poorest people. A limited number of standard medications are available for this disease that has been used for several decades, which have drawbacks such as resistance, higher cost, and patient compliance, making it difficult to reach the poor. The search for novel chemical entities to treat leishmaniasis has led to target-based scaffold research. Thiochromone moieties in conjugation with aromatic amino acids have been considered for the study, along with possible substitutions of the electron-withdrawing and electron-donating groups. N-myristoyl transferase (NMT) has been selected as the molecular target for the study responsible for protein-protein interaction and ribosylation of proteins necessary for the growth inside the human body of the parasite. The designed novel thiochromone analogs were docked against the selected leishmanial NMT using thein-silico methods, physicochemical and toxicity properties were predicted, and Structure-Activity Relationship was also established in-silico. Finally, a molecular dynamics simulation study for 100 ns gave an idea about the stability of the protein-ligand complex. A time frame analysis of each 10 ns confirmation was also studied to understand better the putative binding pattern designed analogs.