mSphere of Influence: Deciphering purine auxotrophy in protozoan parasites.

Bruno Martorelli Di Genova works in parasitology, focusing on Toxoplasma gondii metabolism. In this mSphere of Influence article, he reflects on how the articles "Metabolic Reprogramming during Purine Stress in the Protozoan Pathogen Leishmania donovani" and "Yeast-Based High-Throughput Screen Identifies Plasmodium falciparum Equilibrative Nucleoside Transporter 1 Inhibitors That Kill Malaria Parasites" impacted him, informing his research strategies and understanding of metabolic flexibility in Toxoplasma gondii.

Swarna Bhasma Induces Antigen-Presenting Abilities of Macrophages and Helps Antigen Experienced CD4+ T Cells to Acquire Th1 Phenotypes Against Leishmania donovani Antigens.

In leishmaniasis, the protective immunity is largely mediated by proinflammatory cytokine producing abilities of T cells and an efficient parasite killing by phagocytic cells. Notwithstanding a substantial progress that has been made during last decades, the mechanisms or factors involved in establishing protective immunity against Leishmania are not identified. In ancient Indian literature, metallic "bhasma," particularly that of "swarna" or gold (fine gold particles), is indicated as one of the most prominent metal-based therapeutic medicine, which is known to impart protective and curative properties in various health issues. In this work, we elucidated the potential of swarna bhasma (SB) on the effector properties of phagocytes and antigen-activated CD4+ T cells in augmenting the immunogenicity of L. donovani antigens. The characterization of SB revealing its shape, size, composition, and measurement of cytotoxicity established the physiochemical potential for its utilization as an immunomodulator. The activation of macrophages with SB enhanced their capacity to produce nitric oxide and proinflammatory cytokines, which eventually resulted in reduced uptake of parasites and their proliferation in infected cells. Further, in Leishmania-infected animals, SB administration reduced the generation of IL-10, an anti-inflammatory cytokine, and enhanced pro-inflammatory cytokine generation by antigen activated CD4+ T cells with increased frequency of double (IFNγ+/TNFα+) and triple (IFNγ+TNFα+IL-2+) positive cells and abrogated disease pathogeneses at the early days of infection. Our results also suggested that cow-ghee (A2) emulsified preparation of SB, either alone or with yashtimadhu, a known natural immune modulator which enhances the SB's potential in enhancing the immunogenicity of parasitic antigens. These findings suggested a definite potential of SB in enhancing the effector functions of phagocytes and CD4+ T cells against L. donovani antigens. Therefore, more studies are needed to elucidate the mechanistic details of SB and its potential in enhancing vaccine-induced immunity.

Exploration of potential inhibitors for autophagy-related protein 8 as antileishmanial agents.

Leishmaniasis is considered a tropical neglected disease, which is caused by an intramacrophagic parasite, Leishmania. It is endemic in 89 different countries. Autophagy-related protein 8 (Ldatg8) is responsible for the transformation of parasites from promastigote to amastigote differentiation. Ldatg8 is one of the key drug targets of Leishmania donovani (L. donovani) responsible for the defense of parasites during stress conditions. Virtual screening of natural ligand library had been performed against Ldatg8 to identify novel and potent inhibitors. Molecular docking and molecular dynamics simulation studies showed that urolithin A stably blocked Ldatg8. Urolithins are combinations of coumarin and isocoumarin. Further, we evaluated the antileishmanial effects of urolithin A by antileishmanial assays. Urolithin A inhibited the growth and proliferation of L. donovani promastigotes with an IC50  value of 90.3 ± 6.014 µM. It also inhibited the intramacrophagic parasite significantly with an IC50  value of 78.67 ± 4.62 µM. It showed limited cytotoxicity to the human THP-1 differentiated macrophages with a CC50  value of 190.80 ± 16.89 µM. Further, we assayed reactive oxygen species (ROS) generation and annexin V/PI staining upon urolithin A treatment of parasites to have an insight into the mechanism of its action. It induced ROS significantly in a dose-dependent manner, which caused apoptosis partially in parasites. The potential inhibitors for Ldatg8, identified in this study, would provide the platform for the development of an effective and affordable antileishmanial drug.

Identification of potential antileishmanial 1,3-disubstituted-4-hydroxy-6-methylpyridin-2(1H)-ones, in vitro metabolic stability, cytotoxicity and molecular modeling studies.

We report the synthesis and in vitro evaluation of 1,3-disubstituted-4-hydroxy-6-methylpyridin-2(1H)-one derivatives against Leishmania donovani. Amongst the compound library synthesized, molecules 3d, 3f, 3h, 3i, 3l, and 3m demonstrated substantial dose-dependent killing of the promastigotes. Their IC50 values range from 55.0 to 77.0 µg/ml, with 3m (IC50 55.75 µg/ml) being equipotent with amphotericin B (IC50 50.0 µg/ml, used as standard). The most active compound 3m, is metabolically stable in rat liver microsomes. Furthermore, the molecules are highly specific against leishmania as shown by their weak antibacterial and antifungal activity. In vitro cytotoxicity studies show the compounds lack any cytotoxicity. Furthermore, molecular modeling studies show plausibility of binding to Leishmania donovani topoisomerase 1 (LdTop1). Structure activity relationships reveal bulky substitutions on the pyridone nitrogen are well-tolerated, and such compounds have better binding affinity. Intramolecular hydrogen bonds confer some rigidity to the molecules, rendering a degree of planarity akin to topotecan. Taken together, we emphasis the merits of molecules possessing the 1,3-disubstituted-4-hydroxy-6-methylpyridin-2(1H)-one skeleton as potential antileishmanial agents warranting further investigation.

Case Report: Intravenous Pentamidine Rescue Treatment for Active Chronic Visceral Leishmaniasis in an HIV-1 Infected Patient.

The management of visceral leishmaniasis (VL) in HIV-infected patients is complex because of high mortality rates, toxic drug-related side effects, and a high risk of treatment failure and relapse. We report a case of active chronic VL in an HIV-1-infected woman presenting multiple secondary VL episodes over 7 years leading to massive splenomegaly and blood transfusion-dependent anemia despite several treatment courses and secondary prophylaxis. The patient was finally successfully treated with rescue treatment based on intravenous pentamidine. Twenty months after discontinuation of pentamidine the patient presented complete clinical and parasitological response. In patients with active chronic VL, treatment with intravenous pentamidine can be effective and should be considered as rescue treatment.

Some novel antileishmanial compounds inhibit normal cell cycle progression of Leishmania donovani promastigotes and exhibits pro-oxidative potential.

In the midst of numerous setbacks that beclouds the fight against leishmaniasis; a neglected tropical disease, the search for new chemotherapeutics against this disease is of utmost importance. Leishmaniasis is a disease closely associated with poverty and endemic in Africa, Asia, southern Europe and the Americas. It is caused by parasites of the genus Leishmania and transmitted by a sandfly vector. In this study, we evaluated the antileishmanial potency of eighteen pathogen box compounds and elucidated their biosafety and possible mechanisms of action against Leishmania donovani promastigotes and amastigotes in vitro. IC50s range of 0.12±0.15 to >6.25 µg/ml and 0.13±0.004 to >6.25µg/ml were observed for the promastigotes and amastigotes, respectively. We demonstrated the ability of some of the compounds to cause cytocidal effect on the parasites, induce increased production of reactive oxygen species (ROS), disrupt the normal parasite morphology and cause the accumulation of parasites at the DNA synthesis phase of the cell cycle. We recommend a further in vivo study on these compounds to validate the findings.

Bioflavonoid-Induced Apoptosis and DNA Damage in Amastigotes and Promastigotes of Leishmania donovani: Deciphering the Mode of Action.

Natural products from plants contain many interesting biomolecules. Among them, quercetin (Q), gallic acid (GA), and rutin (R) all have well-reported antileishmanial activity; however, their exact mechanisms of action are still not known. The current study is a step forward towards unveil the possible modes of action of these compounds against Leishmania donovani (the causative agent of visceral leishmaniasis). The selected compounds were checked for their mechanisms of action against L. donovani using different biological assays including apoptosis and necrosis evaluation, effects on genetic material (DNA), quantitative testing of nitric oxide production, ultrastructural modification via transmission electron microscopy, and real-time PCR analysis. The results confirmed that these compounds are active against L. donovani, with IC50 values of 84.65 µg/mL, 86 µg/mL, and 98 µg/mL for Q, GA, and R, respectively. These compounds increased nitric oxide production and caused apoptosis and DNA damage, which led to changes in the treated cells' ultrastructural behavior and finally to the death of L. donovani. These compounds also suppressed essential enzymes like trypanothione reductase and trypanothione synthetase, which are critical for leishmanial survival. The selected compounds have high antileishmanial potentials, and thus in-vivo testing and further screening are highly recommended.

In vitro Leishmanicidal and Trypanosomicidal Properties of Imidazole-Containing Azine and Benzoazine Derivatives.

Leishmaniasis and Chagas diseases are two of the most important parasitic diseases in the world. Both belong to the category of Neglected Tropical Diseases, and they cannot be prevented by vaccination. Their treatments are founded in outdated drugs that possess many pernicious side-effects and they're not easy to administer. With the aim of discovering new compounds that could serve as anti-trypanosomal drugs, an antiparasitic study of a synthetic compound family has been conducted. A series of new 1,4-bis(alkylamino)- and 1-alkylamino-4-chloroazine and benzoazine derivatives 1-4 containing imidazole rings have been synthesized and identified. Their structures showed a possible interest based on previous work. Their in vitro anti-Leishmania infantum, anti-L. braziliensis, anti-L. donovani and anti-T. cruzi activity were tested, as well as the inhibition of Fe-SOD enzymes. It was found that some of them exhibited quite relevant values indicative of being worthy of future more detailed studies, as most of them showed activity to more than only one parasite species, especially compound 3 c was active for the three studied Leishmania species and also for T. cruzi, which is a very interesting trait as it covers a wide spectrum.

Embilica officinalis L. inhibits the growth and proliferation of Leishmania donovani through the induction of ultrastructural changes, mitochondrial dysfunction, oxidative stress and apoptosis-like cell death.

Visceral leishmaniasis (VL) is caused by a protozoan parasite, Leishmania donovani (L. donovani). It affects around 1-2 million people around the world annually. There is an urgent need to investigate new medicament of it due to difficult method of drug administration, long period of treatment, high cost of the drug, adverse side-effects, low efficacy and development of parasite resistance to the available drugs. Medicinal plants have also been used for the treatment of different diseases in traditional system of medicines due to their holistic effects. The Drugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland has already started the program for identification of potential medicinal plant and plant products having antileishmanial potential. Keeping all these in consideration, we planned to study the antileishmanial activity of one of the medicinal plant, Embilica officinalis L. (EO) fruit extract. EO fruit extract inhibited the growth and proliferation of promastigotes as well as intra-macrophagic amastigotes in dose-dependent manner. EO fruit extract induced morphological and ultrastructural changes in parasites as observed under Electron Microscope. It also induced the oxidative stress, mitochondrial dysfunction, DNA laddering and apotosis-like cell death in parasites. Here, we for the first time reported such a detailed mechanism of action of antileishmanial activity of EO fruit extract. Our results suggested that EO fruit extract could be used for the development of new phytomedicine against leishmaniasis.

Utilizing thermal proteome profiling to identify the molecular targets of anti-leishmanial compounds.

Here, we detail our optimized protocol for the identification of drug targets in Leishmania donovani using thermal proteome profiling. This approach is based on the principle that binding of a drug to its protein target can significantly alter the thermal stability of that protein. By monitoring changes in the thermal stability of proteins within drug-treated and untreated cell lysates, using mass spectrometry combined with tandem mass tag labeling, putative targets of the drug can be identified in an unbiased manner. For further details on the use and application of this protocol, please refer to Paradela et al. (2021).

Isolation of Sesquiterpenoids and Steroids from the Soft Coral Sinularia brassica and Determination of Their Absolute Configuration.

Two undescribed rearranged cadinane-type sesquiterpenoids (1-2), named sinulaketol A-B, together with one new chlorinated steroid (3), one new gorgosterol (4), one known sesquiterpene (5), one known dibromoditerpene (6) and two known polyhydroxylated steroids (7-8) were isolated from the soft coral Sinularia brassica. The structures of these compounds were established by extensive spectroscopic analysis, including HRESIMS, 1D, and 2D NMR spectroscopy. Their absolute configurations were also determined by the ECD calculations and DP4+ probability analysis. Antileishmanial activity of compounds 1-8 was evaluated in vitro against the amastigote forms of Leishmania donovani, in which compounds 3, 6, and 7 inhibited the growth of L. donovani by 58.7, 74.3, 54.7%, respectively, at a concentration of 50 µM. Antimicrobial effect of the isolated compounds were also evaluated against Candida albicans, Staphylococcus aureus, and Escherichia coli. Compound 6, a brominated diterpene, exhibited antimicrobial effect against S. aureus.

Pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs as possible candidates against neglected tropical diseases (NTDs): identification of hit compounds towards development of potential treatment of Leishmania donovani.

A rational-based process was adopted for repurposing pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs bearing variable acyl chains, different stereochemical configuration and/or positional relationships. Structural features were highly influential on activity. Amongst, enantiomer 1e having 1,2-vicinal relationship for the -CH2O- and the N-acyl moieties, a saturated palmitoyl chain and an opposite stereochemical configuration to natural sphingolipids was the most potent hit compound against promastigotes showing IC50 value of 28.32 µM. The corresponding enantiomer 1a was 2-fold less potent showing a eudismic ratio of 0.54 in promastigotes. Compounds 1a and 1e inhibited the growth of amastigotes more potently relative to promastigotes. Amongst, enantiomer 1a as the more selective and safer. In silico docking study using a homology model of Leishmania donovani inositol phosphoceramide synthase (IPCS) provided plausible reasoning for the molecular factors underlying the found activity. Collectively, this study suggests compounds 1a and 1e as potential hit compounds for further development of new antileishmanial agents.

Novel azoles with potent antileishmanial activity.

Aim: To investigate the antileishmanial activity of novel azole compounds against Leishmania donovani, which causes deadly visceral leishmaniasis disease. Materials & methods: A focused azole-based library was screened against both promastigotes and amastigotes forms of L. donovani strains in flat-bottomed 96-well tissue culture plates and J774A.1 macrophage cell-line infected with L. donovani. The comprehensive screening of azole-based library against L. donovani strains provided novel hits, which can serve as a good starting point to initiate hit to lead optimization campaign. Results: Hits identified from azole-based library exhibited potent in vitro activity against promastigotes and amastigotes of L. donovani. Conclusion: These potent novel azole hits could be a good starting point to carry out for further medicinal chemistry exploration for antileishmania program.

Boswellic Acids Show In Vitro Activity against Leishmania donovani.

In continuation of our search for leads from medicinal plants against protozoal pathogens, we detected antileishmanial activity in polar fractions of a dichloromethane extract from Boswellia serrata resin. 11-keto-ß-boswellic acid (KBA) could be isolated from these fractions and was tested in vitro against Leishmania donovani axenic amastigotes along with five further boswellic acid derivatives. 3-O-acetyl-11-keto-ß-boswellic acid (AKBA) showed the strongest activity with an IC50 value of 0.88 µM against axenic amastigotes but was inactive against intracellular amastigotes in murine macrophages.

Discovery of Leishmania donovani topoisomerase IB selective inhibitors by targeting protein-protein interactions between the large and small subunits.

Visceral leishmaniasis (VL) is a fatal infectious disease caused by viscerotropic parasitic species of Leishmania. Current treatment options are often ineffective and toxic, and more importantly, there are no clinically validated drug targets available to develop next generation therapeutics against VL. Topoisomerase IB (TopIB) is an essential enzyme for Leishmania survival. The enzyme is organized as a bi-subunit that is distinct from the monomeric topoisomerase I of human. Based on this unique feature, we synthesized peptides composed of partial amino acid sequences of small subunit of Leishmania donovani (Ld) TopIB to confirm a decrease in catalytic activity by interfering the interaction between the two subunits. One of the synthetic peptides, covering essential amino acids for catalytic activity of LdTopIB, interrupted the enzymatic activity. Next, we examined 151 compounds selected from virtual screening in a functional assay and identified three LRL-TP compounds with a significant decrease in LdTopIB activity (IC50 of LRL-TP-85: 1.3 µM; LRL-TP-94: 2.9 µM; and LRL-TP-101: 35.3 µM) and no effects on Homo sapiens (Hs) TopIB activity. Based on molecular docking, the protonated tertiary amine of inhibitors formed key interactions with S415 of the large subunit. The EC50 values of LRL-TP-85, LRL-TP-94, and LRL-TP-101 were respectively 4.9, 1.4, and 27.8 µM in extracellular promastigote assay and 34.0, 53.7, and 11.4 µM in intracellular amastigote assay. Overall, we validated the protein-protein interaction site of LdTopIB as a potential drug target and identified small molecule inhibitors with anti-leishmanial activity.

Biogenic nanoporous silicon carrier improves the efficacy of buparvaquone against resistant visceral leishmaniasis.

Visceral leishmaniasis is a vector-borne protozoan infection that is fatal if untreated. There is no vaccination against the disease, and the current chemotherapeutic agents are ineffective due to increased resistance and severe side effects. Buparvaquone is a potential drug against the leishmaniases, but it is highly hydrophobic resulting in poor bioavailability and low therapeutic efficacy. Herein, we loaded the drug into silicon nanoparticles produced from barley husk, which is an agricultural residue and widely available. The buparvaquone-loaded nanoparticles were several times more selective to kill the intracellular parasites being non-toxic to macrophages compared to the pure buparvaquone and other conventionally used anti-leishmanial agents. Furthermore, the in vivo results revealed that the intraperitoneally injected buparvaquone-loaded nanoparticles suppressed the parasite burden close to 100%. By contrast, pure buparvaquone suppressed the burden only by 50% with corresponding doses. As the conclusion, the biogenic silicon nanoparticles are promising carriers to significantly improve the therapeutic efficacy and selectivity of buparvaquone against resistant visceral leishmaniasis opening a new avenue for low-cost treatment against this neglected tropical disease threatening especially the poor people in developing nations.

Oral combination of eugenol oleate and miltefosine induce immune response during experimental visceral leishmaniasis through nitric oxide generation with advanced cytokine demand.

Conventional therapy of visceral leishmaniasis (VL) remains challenging with the pitfall of toxicity, drug resistance, and expensive. Hence, urgent need for an alternative approach is essential. In this study, we evaluated the potential of combination therapy with eugenol oleate and miltefosine in Leishmania donovani infected macrophages and in the BALB/c mouse model. The interactions between eugenol oleate and miltefosine were found to be additive against promastigotes and amastigotes with xΣFIC 1.13 and 0.68, respectively. Significantly (p < 0.001) decreased arginase activity, increased nitrite generation, improved pro-inflammatory cytokines, and phosphorylated p38MAPK were observed after combination therapy with eugenol oleate and miltefosine. >80% parasite clearance in splenic and hepatic tissue with concomitant nitrite generation, and anti-VL cytokines productions were observed after orally administered miltefosine (5 mg/kg body weight) and eugenol oleate (15 mg/kg body weight) in L. donovani-infected BALB/c mice. Altogether, this study suggested the possibility of an oral combination of miltefosine with eugenol oleate against visceral leishmaniasis.

Oxidant activated soluble adenylate cyclase of Leishmania donovani regulates the cAMP-PKA signaling axis for its intra-macrophage survival during infection.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a stress sensor molecule that transduces the cellular signal when Leishmania donovani moves from insect vector to mammalian host. At this stage, the parasite membrane-bound receptor adenylate cyclase predominantly produces cAMP to cope with the oxidative assault imposed by host macrophages. However, the role of soluble adenylate cyclase of L. donovani (LdHemAC) has not been investigated fully. In the present investigation, we monitored an alternative pool of cAMP, maintained by LdHemAC. The elevated cAMP effectively transmits signals by binding to Protein Kinase A (PKA) present in the cytosol and regulates antioxidant gene expression and phosphorylates several unknown PKA substrate proteins. Menadione-catalyzed production of reactive oxygen species (ROS) mimics host oxidative condition in vitro in parasites where cAMP production and PKA activity were found increased by ~1.54 ± 0.35, and ~1.78 ± 0.47-fold, respectively while expression of LdHemAC gene elevated by ~2.18 ± 0.17-fold. The LdHemAC sense these oxidants and became activated to cyclize ATP to enhance the cAMP basal level that regulates antioxidant gene expression to rescue parasites from oxidative stress. In knockdown parasites (LdHemAC-KD), the downregulated antioxidant genes expression, namely, Sod (2.30 ± 0.46), Pxn (2.73 ± 0.15), Tdr (2.7 ± 0.12), and Gss (1.57 ± 0.15) results in decreased parasite viability while in overexpressed parasites (LdHemAC-OE), the expression was upregulated by ~5.7 ± 0.35, ~2.57 ± 0.56, ~4.7 ± 0.36, and ~2.4 ± 0.83, respectively, which possibly overcomes ROS accumulation and enhances viability. Furthermore, LdHemAC-OE higher PKA activity regulates phosphorylation of substrate proteins (~56 kDs in membrane fraction and ~25 kDs in the soluble fraction). It reduced significantly when treated with inhibitors like DDA, Rp-cAMP, and H-89 and increased by ~2.1 ± 0.28-fold, respectively under oxidative conditions. The LdHemAC-KD was found less infective to RAW 264.7 macrophages and more prone to oxidative damage as compared to LdHemAC-OE and control parasites. Together, this study demonstrates mechanistic links among LdHemAC, cAMP, and PKA in parasite survival and invasion under host oxidative condition.

Synthesis, in vitro antiprotozoal activity, molecular docking and molecular dynamics studies of some new monocationic guanidinobenzimidazoles.

A series of monocationic new guanidinobenzimidazole derivatives were prepared in a four step process starting from 2-nitro-1,4-phenylendiamine. Their antiparasitic activity against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania donovani were evaluated in vitro. Two out of 20 tested monocationic compounds (7, 14) showed close activity with reference drug chloroquine against P. Falciparum. To understand the interactions between DNA minor groove and in vitro active compounds (7, 14) molecular docking studies were carried out. Stability and binding energies of DNA-ligand complexes formed by DNA with compounds 7 and 14 were measured by molecular dynamics simulations throughout 200 ns time. Root mean square deviation (RMSD) values of the ligands remained stable below 0.25 mm and root mean square fluctuation (RMSF) values of the active site residues with which it interacted decreased compared to the apo form. All compounds exhibited theoretical absorption, distribution, metabolism and excretion (ADME) profiles conforming to Lipinski's and Ghose's restrictive rules.

Peptide-mediated leishmaniasis management strategy: Tachyplesin emerges as an effective anti-leishmanial peptide against Leishmania donovani.

Visceral leishmaniasis is one of the neglected tropical diseases caused by an intracellular parasite, Leishmania donovani. Drug resistance, adverse side effects and long treatment regimes are important limitations in achieving the effective elimination of visceral leishmaniasis. In the absence of any vaccine, chemotherapy remains a viable treatment for leishmaniasis. For effective killing of leishmania parasite, the drug molecule needs to cross the cell membrane. In the present study, marine membrane-active peptide Tachyplesin has been used against Leishmania donovani. Further, the mechanism of action and importance of cysteine amino acids of Tachyplesin in anti-leishmanial activity has been assessed. The cargo-carrying ability of Tachyplesin in L. donovani has been established. Thus, dual-use of Tachyplesin as an anti-leishmanial peptide as well as a cargo delivery vehicle makes the marine peptide an attractive therapeutic target against visceral leishmaniasis.