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Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. Current therapies are unsuitable, and there is an urgent need for safe, short-course, and low-cost oral treatments to combat this neglected disease. The benzoxaborole chemotype has previously delivered clinical candidates for the treatment of other parasitic diseases. Here, we describe the development and optimization of this series, leading to the identification of compounds with potent in vitro and in vivo antileishmanial activity. The lead compound (DNDI-6148) combines impressive in vivo efficacy (>98% reduction in parasite burden) with pharmaceutical properties suitable for onward development and an acceptable safety profile. Detailed mode of action studies confirm that DNDI-6148 acts principally through the inhibition of Leishmania cleavage and polyadenylation specificity factor (CPSF3) endonuclease. As a result of these studies and its promising profile, DNDI-6148 has been declared a preclinical candidate for the treatment of VL.

The phosphodiesterase inhibitor tetrahydrophthalazinone NPD-008 was explored by phenotypic in vitro screening, target validation, and ultrastructural approaches against Trypanosoma cruzi . NPD-008 displayed activity against different forms and strains of T. cruzi (50% effective concentration [EC 50 ], 6.6 to 39.5 μM). Increased cAMP levels of T. cruzi and its combination with benznidazole gave synergistic interaction.

As there is a continuous need for novel anti-infectives, the present study aimed to fuse two modes of action into a novel 3-nitroimidazo[1,2-b]pyridazine scaffold to improve antiparasitic efficacy. For this purpose, we combined known structural elements of phosphodiesterase inhibitors, a target recently proposed for Trypanosoma brucei and Giardia lamblia, with a nitroimidazole scaffold to generate nitrosative stress. The compounds were evaluated in vitro against a panel of protozoal parasites, namely Giardia lamblia, Trypanosoma brucei, T. cruzi, Leishmania infantum and Plasmodium falciparum and for cytotoxicity on MRC-5 cells. Interestingly, selective sub-nanomolar activity was obtained against G. lamblia, and by testing several analogues with and without the nitro group, it was shown that the presence of a nitro group, but not PDE inhibition, is responsible for the low IC50 values of these novel compounds. Adding the favourable drug-like properties (low molecular weight, cLogP (1.2–4.1) and low polar surface area), the key compounds from the 3-nitroimidazo[1,2-b]pyridazine series can be considered as valuable hits for further anti-giardiasis drug exploration and development.

Dry eye syndrome (DES), a multifactorial disorder which leads to ocular discomfort, visual disturbance and tear film instability, has a rising prevalence and limited treatment options. In this study, a newly developed trypsin-like serine protease inhibitor (UAMC-00050) in a tear drop formulation was evaluated to treat ocular inflammation. A surgical animal model of dry eye was employed to investigate the potential of UAMC-00050 on dry eye pathology. Animals treated with UAMC-00050 displayed a significant reduction in ocular surface damage after evaluation with sodium fluorescein, compared to untreated, vehicle treated and cyclosporine-treated animals. The concentrations of IL-1α and TNF-α were also significantly reduced in tear fluid from UAMC-00050-treated rats. Additionally, inflammatory cell infiltration in the palpebral conjunctiva (CD3 and CD45), was substantially reduced. An accumulation of pro-MMP-9 and a decrease in active MMP-9 were found in tear fluid from animals treated with UAMC-00050, suggesting that trypsin-like serine proteases play a role in activating MMP-9 in ocular inflammation in this animal model. Comparative qRT-PCR analyses on ocular tissue indicated the upregulation of tryptase, urokinase plasminogen activator receptor (uPAR) and protease-activated receptor 2 (PAR2). The developed UAMC-00050 formulation was stable up to 6 months at room temperature in the absence of light, non-irritating and sterile with compatible pH and osmolarity. These results provide a proof-of-concept for the in vivo modifying potential of UAMC-00050 on dry eye pathology and suggest a central role of trypsin-like serine proteases and PAR2 in dry eye derived ocular inflammation. ispartof: Scientific Reports vol:10 issue:1 ispartof: location:England status: published

Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of Africa. In this manuscript we describe the optimization of a family of phtalazinone derivatives. Phosphodiesterases have emerged as attractive molecular targets for a novel treatment for a variety of neglected parasitic diseases. Compound 1 resulted in being a potent TbrPDEB1 inhibitor with interesting activity against T. brucei in a phenotypic screen. Derivative 1 was studied in an acute in vivo mouse disease model but unfortunately showed no efficacy due to low metabolic stability. We report structural modifications to achieve compounds with an improved metabolic stability while maintaining high potency against TbrPDEB1 and T. brucei. Compound 14 presented a good microsomal stability in mouse and human microsomes and provides a good starting point for future efforts.

© The Author(s) 2020., [Background]: Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure., [Methods]: Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis., [Results]: Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector., [Conclusions]: Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector., Funding was available through the Research Fund Flanders (G051812N, 12I0317N and 1189617N) and a research fund of the University of Antwerp (TT-ZAPBOF 33049 and TOP-BOF 35017). LR was supported by Redes de Investigación Cooperativa RETICS-FEDER RD16/0027/0010.

African trypanosomiasis is a disease caused by Trypanosoma brucei parasites with limited treatment options. Trypanosoma is unable to synthesize purines de novo and relies solely on their uptake and interconversion from the host, constituting purine nucleoside analogues a potential source of antitrypanosomal agents. Here we combine structural elements from known trypanocidal nucleoside analogues to develop a series of 3’-deoxy-7-deazaadenosine nucleosides, and investigate their effects against African trypanosomes. 3’-Deoxytubercidin is a highly potent trypanocide in vitro and displays curative activity in animal models of acute and CNS-stage disease, even at low doses and oral administration. Whole-genome RNAi screening reveals that the P2 nucleoside transporter and adenosine kinase are involved in the uptake and activation, respectively, of this analogue. This is confirmed by P1 and P2 transporter assays and nucleotide pool analysis. 3’-Deoxytubercidin is a promising lead to treat late-stage sleeping sickness., Trypanosoma brucei relies on uptake and conversion of purines from the host, which constitutes a potential drug target. Here, Hulpia et al. combine structural elements from known trypanocidal nucleoside analogues and develop a potent trypanocide with curative activity in animal models of acute and late stage sleeping sickness.

Several 3',5'-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity. ispartof: BIOORGANIC & MEDICINAL CHEMISTRY vol:27 issue:18 pages:3998-4012 ispartof: location:England status: published

9 p.-5 fig.-2 tab., We report the evaluation of 265 compounds from a PDE-focused library for their antischistosomal activity, assessed in vitro using Schistosoma mansoni. Of the tested compounds, 171 (64%) displayed selective in vitro activity, with 16 causing worm hypermotility/spastic contractions and 41 inducing various degrees of worm killing at 100 μM, with the surviving worms displaying sluggish movement, worm unpairing and complete absence of eggs. The compounds that did not affect worm viability (n=72) induced a complete cessation of ovipositing. 82% of the compounds had an impact on male worms whereas female worms were barely affected. In vivo evaluation in S. mansoni-infected mice with the in vitro ‘hit’ NPD-0274 at 20 mg/kg/day orally for 5 days resulted in worm burden reductions of 29% and intestinal tissue egg load reduction of 35% at 10 days posttreatment.Combination of praziquantel (PZQ) at 10 mg/kg/day for 5 days with NPD-0274 or NPD-0298 resulted in significantly higher worm killing than PZQ alone, as well as a reduction in intestinal tissue egg load, disappearance of immature eggs and an increase in the number of dead eggs., This work is part of the PDE4NPD consortium supported by Framework Program 7 of the European Commission No: 602666.Funding from RICET/FEDER funds (RD16/0027/0010), and the Ministry of Education, Culture and Sport (MECD) of Spain (Grant FPU15/1465 to V. S.-P.) is also acknowledged. Y. Z. was supported by a grant of the Chinese Science Council; A. R. B. and I. J. P. dE. were supported by the Netherlands Science Foundation.

Chagas disease and visceral leishmaniasis are two neglected tropical diseases responsible for numerous deaths around the world. For both, current treatments are largely inadequate, resulting in a continued need for new drug discovery. As both kinetoplastid parasites are incapable of

Ethnopharmacological relevance Terminalia albida (Combretaceae), widely used in Guinean traditional medicine, showed promising activity against Plasmodium falciparum and Candida albicans in previous studies. Bioassay-guided fractionation was carried out in order to isolate the compounds responsible for these activities. Materials and methods Fractionation and isolation were performed by flash chromatography, followed by semi-preparative HPLC-DAD-MS. The structural elucidation of the isolated compounds was carried out by 1D and 2D NMR as well as HR-ESI-MS. Isolated compounds were evaluated against Plasmodium falciparum, Candida albicans, Staphylococcus aureus and Escherichia coli, and their cytotoxicity against MRC-5 cells was determined. Results Bioassay-guided fractionation of Terminalia albida root resulted in the isolation of 14 compounds (1–14), and their antimicrobial properties were evaluated. Pantolactone (1) (IC50 0.60 ± 0.03 μM) demonstrated significant activity against P. falciparum. Other compounds, including 3,4,3′-tri-O-methyl-ellagic acid (3), the triterpenes arjunolic acid (5), arjungenin (6), arjunic acid (7) and arjunglucoside II (10), and the phenol glycoside calophymembranside-B (14), were less active and showed IC50 values in the range 5–15 μM. None of the tested compound showed antibacterial or antifungal activity. Conclusion These results may explain at least in part the activity of the root extract of T. albida against P. falciparum.

Kinetoplastid parasites are the causative agents of neglected tropical diseases with an unmet medical need. These parasites are unable to synthesize the purine ring de novo, and therefore rely on purine salvage to meet their purine demand. Evaluating purine nucleoside analogs is therefore an attractive strategy to identify antikinetoplastid agents. Several anti-Trypanosoma cruzi and anti-Trypanosoma brucei 7-deazapurine nucleosides were previously discovered, with the removal of the 3'-hydroxyl group resulting in a significant boost in activity. In this work we therefore decided to assess the effect of the introduction of a 3'-fluoro substituent in 7-deazapurine nucleosides on the anti-kinetoplastid activities. Hence, we synthesized two series of 3'-deoxy-3'-fluororibofuranosyl and 3'-deoxy-3'-fluoroxylofuranosyl nucleosides comprising 7-deazaadenine and -hypoxanthine bases and assayed these for antiparasitic activity. Several analogs with potent activity against T. cruzi and T. brucei were discovered, indicating that a fluorine atom in the 3'-position is a promising modification for the discovery of anti-parasitic nucleosides. (C) 2021 Elsevier Masson SAS. All rights reserved.

Neglected parasitic diseases remain a major public health issue worldwide, especially in tropical and subtropical areas. Human parasite diversity is very large, ranging from protozoa to worms. In most cases, more effective and new drugs are urgently needed. Previous studies indicated that the gold(I) drug auranofin (Ridaura&reg, ) is effective against several parasites. Among new gold(I) complexes, the phosphole-containing gold(I) complex {1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl (abbreviated as GoPI) is an irreversible inhibitor of both purified human glutathione and thioredoxin reductases. GoPI-sugar is a novel 1-thio-&beta, d-glucopyranose 2,3,4,6-tetraacetato-S-derivative that is a chimera of the structures of GoPI and auranofin, designed to improve stability and bioavailability of GoPI. These metal-ligand complexes are of particular interest because of their combined abilities to irreversibly target the essential dithiol/selenol catalytic pair of selenium-dependent thioredoxin reductase activity, and to kill cells from breast and brain tumors. In this work, screening of various parasites&mdash, protozoans, trematodes, and nematodes&mdash, was undertaken to determine the in vitro killing activity of GoPI-sugar compared to auranofin. GoPI-sugar was found to efficiently kill intramacrophagic Leishmania donovani amastigotes and adult filarial and trematode worms.

Neglected tropical diseases remain among the most critical public health concerns in Africa and South America. The drug treatments for these diseases are limited, which invariably leads to fatal cases. Hence, there is an urgent need for new antitrypanosomal drugs. To address this issue, a large number of diverse heterocyclic compounds were prepared. Straightforward synthetic approaches tolerated pre‐functionalized structures, giving rise to a structurally diverse set of analogs. We report on a set of 57 heterocyclic compounds with selective activity potential against kinetoplastid parasites. In general, 29 and 19 compounds of the total set could be defined as active against Trypanosoma cruzi and T. brucei brucei, respectively (antitrypanosomal activities, Attention to the neglected: In this study we modified imidazopyridine/pyrimidine and furopyridine cores at eight different positions to obtain various heterocyclic compounds as anti‐infective agents. This work explores the structure−activity relationships against various Trypanosoma subtypes. These imidazopyridine/pyrimidine‐ and furopyridine‐based compounds demonstrate high antitrypanosomal activities on parasite cultures and show significant promise for trypanosomiases drug discovery.

Previous investigation of the potent antileishmanial properties of antitubercular 7-substituted 2-nitroimidazo[2,1-b][1,3]oxazines with biaryl side chains led to our development of a new clinical candidate for visceral leishmaniasis (DNDI-0690). Within a collaborative backup program, a racemic monoaryl lead (3) possessing comparable activity in mice but a greater hERG liability formed the starting point for our pursuit of efficacious second generation analogues having good solubility and safety. Asymmetric synthesis and appraisal of its enantiomers first established that chiral preferences for in vivo efficacy were species dependent and that neither form afforded a reduced hERG risk. However, in line with our findings in a structurally related series, less lipophilic heteroaryl ethers provided significant solubility enhancements (up to 16-fold) and concomitantly attenuated hERG inhibition. One promising pyridine derivative (49) displayed 100% oral bioavailability in mice and delivered a 96% parasite burden reduction when dosed at 50 mg/kg in a Leishmania donovani mouse model of visceral leishmaniasis.

Human African trypanosomiasis is a neglected tropical disease caused by

Ethnopharmacological relevance In Guinea, medicinal plants play an important role in the management of infectious diseases including urinary disorders, skin diseases and oral diseases. This study was carried out to collect medicinal plant species employed for the treatment of these diseases and to investigate their antimicrobial potential. Materials and methods Based on an ethnobotanical investigation carried out in three Guinean regions, 74 traditional healers and 28 herbalists were interviewed and medicinal plants were collected. The most quoted plant species were evaluated for their antimicrobial activities against Staphylococcus aureus, Escherichia coli, Candida albicans, and in addition against Plasmodium falciparum. Results A total of 112 plant species belonging to 102 genera distributed over 42 botanical families were inventoried. Among the selected plant species, promising activities against C. albicans were obtained for the methanolic extracts of the stem bark of Terminalia albida (IC50 1.2 μg/ml), the leaves of Tetracera alnifolia (IC50 1.6 μg/ml) and the root bark of Swartzia madagascariensis (IC50 7.8 μg/ml). The highest activity against S. aureus was obtained for the dichloromethane extracts of the leaves of Pavetta crassipes (IC50 8.5 μg/ml) and the root of Swartzia madagascariensis (IC50 12.8 μg/ml). Twenty one extracts, obtained from twelve plant species, were strongly active against Plasmodium falciparum, including the dichloromethane extracts of the root and stem bark of Terminalia albida root (IC50 0.6 and 0.8 μg/ml), the leaves of Landolphia heudelotii (IC50 0.5 μg/ml), the stem bark of Combretum paniculatum (IC50 0.4 μg/ml) and the leaves of Gardenia ternifolia (IC50 1.3 μg/ml). Conclusion The present study provides a comprehensive overview of medicinal plants employed by Guinean traditional healers for the treatment of various microbial diseases, including urinary disorders, skin diseases and oral diseases. Some of the studied plant species showed promising antimicrobial activity and could be considered as a potential source for the development of new antifungal and/or antimalarial agents.

In vitro growth (inhibition) assays have a dual application, either supporting the discovery of novel drugs or as a monitoring tool of drug resistance in patient isolates. From an experimental design point of view, both are quite different with regard to the infecting Leishmania species and strain, the wide variety of permissive host cells (primary cells versus cell lines), drug exposure times, detection methods and endpoint criteria. Recognizing the need for enhanced assay standardization to decrease interlaboratory variation and improve proper interpretation of results, a detailed description is given of the basic fundamental procedures and requirements for routine in vitro growth of Leishmania spp. with specific focus on the intracellular amastigote susceptibility assay. Although the described experimental procedures focus on visceral Leishmania species, the same assay principles may apply for the cutaneous species as well.

Background Molecular detection techniques using peripheral blood are preferred over invasive tissue aspiration for the diagnosis and post-treatment follow-up of visceral leishmaniasis (VL) patients. This study aims to identify suitable stabilizing reagents to prevent DNA and RNA degradation during storage and transport to specialized laboratories where molecular diagnosis is performed. Methodology The stabilizing capacities of different commercially available reagents were compared using promastigote-spiked human blood and peripheral blood of Syrian golden hamsters subjected to experimental infection, treatment (miltefosine or aminopyrazole DNDi-1044) and immunosuppression. The impact of various storage temperature conditions was tested in combination with an established kinetoplast DNA (kDNA) qPCR and a recently developed spliced leader RNA (SL-RNA) assay for Leishmania detection. Principal findings Irrespective of the blood type and stabilizer used, threshold (cT) values obtained with the SL-RNA qPCR were systematically lower than those obtained with the kDNA assay, confirming the advantage of the SL-RNA assay over the widely used kDNA assay for low-level Leishmania detection. Peripheral blood parasite levels correlated relatively well with hepatic burdens. RNA protect cell reagent provided the most optimal simultaneous DNA and RNA stabilization in both human and hamster blood. However, this stabilizer requires an erythrocyte lysis step, which can be challenging under field conditions. DNA/RNA shield provides a good alternative for downstream kDNA and SL-RNA assays, especially if sample storage capacity at 4 °C can be guaranteed. Conclusions/Significance The recommended stabilizing reagents are compatible with RNA- and DNA-based Leishmania detection in peripheral blood in the VL hamster model and spiked human blood. Since molecular detection techniques using peripheral blood are less invasive than microscopic assessment of tissue aspirates, the findings of this study may be applied to human VL clinical studies.

For the development of new and potent antimalarial drugs, we designed the virtual library with three points of randomization of novel [1,2,4]triazolo[4,3-a]pyridines bearing a sulfonamide fragment. The library of 1561 compounds has been investigated by both virtual screening and molecular docking methods using falcipain-2 as a target enzyme. 25 chosen hits were synthesized and evaluated for their antimalarial activity in vitro against Plasmodium falciparum. 3-Ethyl-N-(3-fluorobenzyl)-N-(4-methoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine-6-sulfonamide and 2-(3-chlorobenzyl)-8-(piperidin-1-ylsulfonyl)-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one showed in vitro good antimalarial activity with inhibitory concentration IC50 = 2.24 and 4.98 &mu, M, respectively. This new series of compounds may serve as a starting point for future antimalarial drug discovery programs.

The present study aimed to develop, characterize and evaluate the amphotericin B–loaded nanostructured lipid carriers (AmB-NLCs) for topical treatment of cutaneous leishmaniasis (CL) and vulvovaginal candidiasis (VVC). AmB-NLCs were characterized for particle size, zeta potential, encapsulation efficiency and surface morphology. Prepared NLCs were also characterized for in vitro drug release, ex vivo skin permeation and deposition before evaluating their in vitro and in vivo efficacy. Cytotoxicity of NLCs was assessed on MRC-5 cells, whereas skin irritation potential was evaluated in vivo using rats. Significant accumulation of drug in to the skin supported the topical application potential of drug-loaded NLCs. Encapsulation of AmB in NLCs resulted in enhanced in vitro potency against promastigotes and intracellular amastigotes of L. major JISH 118 (IC50 ± SEM = 0.02 ± 0.1 μM for both) compared with free drug (IC50 ± SEM = 0.15 ± 0.2 & 0.14 ± 0.0, respectively). Similar improved potency of AmB-NLCs was also observed for other Leishmania and fungal strains compared with drug solution. Topical application of AmB-NLCs on L. major–infected BALB/c mice caused a significant reduction in parasite burden per mg of lesion (65 × 108 ± 13) compared with the control group (> 167.8 × 108 ± 11). Topical AmB-NLCs gel demonstrated superior efficacy in the vaginal C. albicans rat model for VVC as compared with plain AmB gel. Moreover, results of in vitro cytotoxicity assay and in vivo skin irritation test confirmed AmB-NLCs to be non-toxic and safe for topical use. In conclusion, NLCs may have promising potential as carrier for topical treatment of various conditions of skin and mucosa.

African trypanosomiasis, a deadly infectious disease caused by the protozoan Trypanosoma brucei spp., is spread to new hosts by bites of infected tsetse flies. Currently approved therapies all have their specific drawbacks, prompting a search for novel therapeutic agents. T. brucei lacks the enzymes necessary to forge the purine ring from amino acid precursors, rendering them dependent on the uptake and interconversion of host purines. This dependency renders analogues of purines and corresponding nucleosides an interesting source of potential anti-T. brucei agents. In this study, we synthesized and evaluated a series of 7-substituted 7-deazainosine derivatives and found that 6-O-alkylated analogues in particular showed highly promising in vitro activity with EC50 values in the mid-nanomolar range. SAR investigation of the O-alkyl chain showed that antitrypanosomal activity increased, and also cytotoxicity, with alkyl chain length, at least in the linear alkyl chain series. However, this could be attenuated by introducing a terminal branch point, resulting in the highly potent and selective analogues, 36, 37 and 38. No resistance related to transporter-mediated uptake could be identified, earmarking several of these analogues for further in vivo follow-up studies. (C) 2020 Elsevier Masson SAS. All rights reserved.

Leishmania parasites have the capacity to rapidly adapt to changing environments in their digenetic life cycle which alternates between a vertebrate and an invertebrate host. Emergence of resistance following drug exposure can evoke phenotypic alterations that affect several aspects of parasite fitness in both hosts. Current studies of the impact of resistance are mostly limited to interactions with the mammalian host and characterization of in vitro parasite growth and differentiation. Development in the vector and transmission capacity have been largely ignored. This review reflects on the impact of drug resistance on its spreading potential with specific focus on the use of the sand fly infection model to evaluate parasite development in the vector and the ensuing transmission potential of drug-resistant phenotypes.

Ferroptosis is an iron-catalysed, non-apoptotic form of regulated necrosis that results in oxidative lipid damage in cell membranes that can be inhibited by the radical-trapping antioxidant Ferrostatin-1 (Fer-1). Novel inhibitors derived from the Fer-1 scaffold inhibited ferroptosis potently but suffered from solubility issues. In this paper, we report the synthesis of a more stable and readily soluble series of Fer-1 analogues that potently inhibit ferroptosis. The most promising compounds (37, 38 and 39) showed an improved protection compared to Fer-1 against multi-organ injury in mice. No toxicity was observed in mice after daily injection of 39 (UAMC-3203) for 4 weeks. UAMC-3203 inserts rapidly in a phospholipid bilayer in silico, which aligns with the current understanding of the mechanism of action of these compounds. Concludingly, these analogues have superior properties compared to Fer-1, show in vivo efficacy and represent novel lead compounds with therapeutic potential in relevant ferroptosis-driven disease models.

Chagas disease is the leading cause of cardiac-related mortality in Latin American countries where it is endemic. Trypanosoma cruzi, the disease-causing pathogen, is unable to synthesize purines de novo, necessitating salvage of preformed host purines. Therefore, purine and purine-nucleoside analogues might constitute an attractive source for identifying antitrypanosomal hits. In this study, structural elements of two purine-nucleoside analogues (i.e., cordycepin and a recently discovered 7-substituted 7-deazaadenosine) led to the identification of novel nucleoside analogues with potent in vitro activity. The structureactivity relationships of substituents at C-7 were investigated, ultimately leading to the selection of compound 5, with a C-7 para-chlorophenyl group, for in vivo evaluation. This derivative showed complete suppression of T. cruzi Y-strain blood parasitemia when orally administered twice daily for 5 days at 25 mg/kg and was able to protect infected mice from parasite-induced mortality. However, sterile cure by immunosuppression could not be demonstrated.

Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of sub-saharan Africa. There is a high unmet medical need since the approved drugs are poorly efficacious, show considerable toxicity and are not easy to administer. This work describes the optimization of the pharmacokinetic properties of a previously published family of triazine lead compounds. One compound (35 (UAMC-03011)) with potent anti-trypanosomal activity and no cytotoxicity was selected for further study because of its good microsomal stability and high selectivity for Trypanosoma brucei over a panel including Trypanosoma cruzi, L.eishmania infantum, and Plasmodium falciparum. In vivo pharmacokinetic parameters were determined and the compound was studied in an acute in vivo mouse disease model. One of the important learnings of this study was that the rate of trypanocidal activity is an important parameter during the lead optimization process.

Several B. cenocepacia mouse models are available to study the pulmonary infection by this Burkholderia cepacia complex (BCC) species. However, a characterized B. cenocepacia mouse model to evaluate the efficacy of potential new antibacterial therapies is not yet described. Therefore, we optimized and validated the course of infection (Le. bacterial proliferation in lung, liver and spleen) and the efficacy of a reference antibiotic, tobramycin (TOB), in a mouse lung infection model. Furthermore, the local immune response and histological changes in lung tissue were studied during infection and treatment. A reproducible lung infection was observed when immunosuppressed BALB/c mice were infected with B. cenocepacia LMG 16656. Approximately 50 to 60% of mice infected with this BCC species demonstrated a dissemination to liver and spleen. TOB treatment resulted in a two log reduction in lung burden, prevented dissemination of B. cenocepacia to liver and spleen and significantly reduced levels of proinflammatory cytokines. As this mouse model is characterized by a reproducible course of infection and efficacy of TOB, it can be used as a tool for the in vivo evaluation of new antibacterial therapies.

Background Host proteases have been shown to play important roles in many viral activities such as entry, uncoating, viral protein production and disease induction. Therefore, these cellular proteases are putative targets for the development of antivirals that inhibit their activity. Host proteases have been described to play essential roles in Ebola, HCV, HIV and influenza, such that specific protease inhibitors are able to reduce infection. RSV utilizes a host protease in its replication cycle but its potential as antiviral target is unknown. Therefore, we evaluated the effect of protease inhibitors on RSV infection. Methods To measure the sensitivity of RSV infection to protease inhibitors, cells were infected with RSV and incubated for 18 h in the presence or absence of the inhibitors. Cells were fixed, stained and studied using fluorescence microscopy. Results Several protease inhibitors, representing different classes of proteases (AEBSF, Pepstatin A, E-64, TPCK, PMSF and aprotinin), were tested for inhibitory effects on an RSV A2 infection of HEp-2 cells. Different treatment durations, ranging from 1 h prior to inoculation and continuing for 18 h during the assay, were evaluated. Of all the inhibitors tested, AEBSF and TPCK significantly decreased RSV infection. To ascertain that the observed effect of AEBSF was not a specific feature related to HEp-2 cells, A549 and BEAS-2B cells were also used. Similar to HEp-2, an almost complete block in the number of RSV infected cells after 18 h of incubation was observed and the effect was dose-dependent. To gain insight into the mechanism of this inhibition, AEBSF treatment was applied during different phases of an infection cycle (pre-, peri- and post-inoculation treatment). The results from these experiments indicate that AEBSF is mainly active during the early entry phase of RSV. The inhibitory effect was also observed with other RSV isolates A1998/3–2 and A2000/3–4, suggesting that this is a general feature of RSV. Conclusion RSV infection can be inhibited by broad serine protease inhibitors, AEBSF and TPCK. We confirmed that AEBSF inhibition is independent of the cell line used or RSV strain. The time point at which treatment with the inhibitor was most potent, was found to coincide with the expected moment of entry of the virion with the host cell.

Respiratory Syncytial Virus (RSV) is a very important viral pathogen in children, immunocompromised and cardiopulmonary diseased patients and the elderly. Most of the published research with RSV was performed on RSV Long and RSV A2, isolated in 1956 and 1961, yet recent RSV isolates differ from these prototype strains. Additionally, these viruses have been serially passaged in cell culture, which may result in adaptations that affect virus&ndash, host interactions. We have isolated RSV from mucosal secretions of 12 patients in the winters 2016&ndash, 2017 and 2017&ndash, 2018, of which eight RSV-A subtypes and four RSV-B subtypes. Passage 3 of the isolates was assessed for viral replication kinetics and infectious virus production in HEp-2, A549 and BEAS-2B cells, thermal stability at 37 &deg, C, 32 &deg, C and 4 &deg, C, syncytia formation, neutralization by palivizumab and mucin mRNA expression in infected A549 cells. We observed that viruses isolated in one RSV season show differences on the tested assays. Furthermore, comparison with RSV A2 and RSV B1 reveals for some RSV isolates differences in viral replication kinetics, thermal stability and fusion capacity. Major differences are, however, not observed and differences between the recent isolates and reference strains is, overall, similar to the observed variation in between the recent isolates. One clinical isolate (BE/ANT-A11/17) replicated very efficiently in all cell lines, and remarkably, even better than RSV A2 in the HEp-2 cell line.

The structure-activity relationship of a 4-Azaindole-2-piperidine compound selected from GlaxoSmithKline’s recently disclosed open-resource “Chagas box” and possessing moderate activity against Trypanosoma cruzi, the parasite responsible for Chagas disease, is presented. Despite considerable medicinal chemistry efforts, a suitably potent and metabolically stable compound could not be identified to advance the series into in vivo studies. This research should be of interest to those in the area of neglected diseases and in particular anti-kinetoplastid drug discovery.

Background Chagas’ disease, caused by the protozoan parasite Trypanosoma cruzi, needs urgent alternative therapeutic options as the treatments currently available display severe limitations, mainly related to efficacy and toxicity. Objectives As phosphodiesterases (PDEs) have been claimed as novel targets against T. cruzi, our aim was to evaluate the biological aspects of 12 new phthalazinone PDE inhibitors against different T. cruzi strains and parasite forms relevant for human infection. Methods In vitro trypanocidal activity of the inhibitors was assessed alone and in combination with benznidazole. Their effects on parasite ultrastructural and cAMP levels were determined. PDE mRNA levels from the different T. cruzi forms were measured by quantitative reverse transcription PCR. Results Five TcrPDEs were found to be expressed in all parasite stages. Four compounds displayed strong effects against intracellular amastigotes. Against bloodstream trypomastigotes (BTs), three were at least as potent as benznidazole. In vitro combination therapy with one of the most active inhibitors on both parasite forms (NPD-040) plus benznidazole demonstrated a quite synergistic profile (xΣ FICI = 0.58) against intracellular amastigotes but no interaction (xΣ FICI = 1.27) when BTs were assayed. BTs treated with NPD-040 presented disrupted Golgi apparatus, a swollen flagellar pocket and signs of autophagy. cAMP measurements of untreated parasites showed that amastigotes have higher ability to efflux this second messenger than BTs. NPD-001 and NPD-040 increase the intracellular cAMP content in both BTs and amastigotes, which is also released into the extracellular milieu. Conclusions The findings demonstrate the potential of PDE inhibitors as anti-T. cruzi drug candidates.

Inhibitors against Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) and B2 (TbrPDEB2) have gained interest as new treatments for human African trypanosomiasis. The recently reported alkynamide tetrahydrophthalazinones, which show submicromolar activities against TbrPDEB1 and anti-T. brucei activity, have been used as starting point for the discovery of new TbrPDEB1 inhibitors. Structure-based design indicated that the alkynamide-nitrogen atom can be readily decorated, leading to the discovery of 37, a potent TbrPDEB1 inhibitor with submicromolar activities against T. brucei parasites. Furthermore, 37 is more potent against TbrPDEB1 than hPDE4 and shows no cytotoxicity on human MRC-5 cells. The crystal structures of the catalytic domain of TbrPDEB1 co-crystalized with several different alkynamides show a bidentate interaction with key-residue Gln874, but no interaction with the parasite-specific P-pocket, despite being (uniquely) a more potent inhibitor for the parasite PDE. Incubation of blood stream form trypanosomes by 37 increases intracellular cAMP levels and results in the distortion of the cell cycle and cell death, validating phosphodiesterase inhibition as mode of action. ispartof: BIOORGANIC & MEDICINAL CHEMISTRY vol:27 issue:18 pages:4013-4029 ispartof: location:England status: published

Seven tetracyclic spiro-alkaloids, i.e. glucoerysodine (

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization worldwide each year and there is presently no licensed vaccine to prevent severe RSV infections. Two major RSV glycoproteins, attachment (G) and fusion (F) protein, regulate viral replication and both proteins contain potential glycosylation sites which are highly variable for the G protein and conserved for the F protein among virus isolates. The RSV F sequence possesses five N-glycosylation sites located in the F2 subunit (N27 and N70), the p27 peptide (N116 and N126) and the F1 subunit (N500). The importance of RSV F N-glycosylation in virus replication and immunogenicity is not yet fully understood, and a better understanding may provide new insights for vaccine development. By using a BAC-based reverse genetics system, recombinant viruses expressing F proteins with loss of N-glycosylation sites were made. Mutant viruses with single N-glycosylation sites removed could be recovered, while this was not possible with the mutant with all N-glycosylation sites removed. Although the individual RSV F N-glycosylation sites were shown not to be essential for viral replication, they do contribute to the efficiency of in vitro and in vivo viral infection. To evaluate the role of N-glycosylation sites on RSV F antigenicity, serum antibody titers were determined after infection of BALB/c mice with RSV expressing the glycomutant F proteins. Infection with recombinant virus lacking the N-glycosylation site at position N116 (RSV F N116Q) resulted in significant higher neutralizing antibody titers compared to RSV F WT infection, which is surprising since this N-glycan is present in the p27 peptide which is assumed to be absent from the mature F protein in virions. Thus, single or combined RSV F glycomutations which affect virus replication and fusogenicity, and which may induce enhanced antibody responses upon immunization could have the potential to improve the efficacy of RSV LAV approaches.

Leishmaniasis is a neglected parasitic disease for which the current antileishmania therapeutics are hampered by drug toxicity, high cost, need for parenteral administration, increasing treatment failure rates, and emergence of drug resistance. The R&D pipeline had run fairly dry for several years, but fortunately some new drug candidates are now under (pre)clinical development. Identification of novel drugs will nevertheless remain essential to adequately sustain and improve effective disease control in the future. In this review, a package of standard and accessible R&D approaches is discussed with expansion to some alternative strategies focusing on parasite-host and vector-host interactions.

Chagas disease is becoming a worldwide problem; it is currently estimated that over six million people are infected. The two drugs in current use, benznidazole and nifurtimox, require long treatment regimens, show limited efficacy in the chronic phase of infection, and are known to cause adverse effects. Phenotypic screening of an in-house library led to the identification of 2,2′-methylenebis(5-(4-bromophenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one), a phenyldihydropyrazolone dimer, which shows an in vitro pIC50 value of 5.4 against Trypanosoma cruzi. Initial optimization was done by varying substituents of the phenyl ring, after which attempts were made to replace the phenyl ring. Finally, the linker between the dimer units was varied, ultimately leading to 2,2′-methylenebis(5-(3-bromo-4-methoxyphenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (NPD-0228) as the most potent analogue. NPD-0228 has an in vitro pIC50 value of 6.4 against intracellular amastigotes of T. cruzi and no apparent toxicity against the human MRC-5 cell line and murine cardiac cells.

Fosmidomycin is a natural antibiotic with promising IspC (DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase) inhibitory activity. This enzyme catalyzes the first committed step of the non-mevalonate isoprenoid biosynthesis pathway, which is essential in Plasmodium falciparum and Mycobacterium tuberculosis. Mainly as a result of its high polarity, fosmidomycin displays suboptimal pharmacokinetic properties. Furthermore, fosmidomycin is inactive against M. tuberculosis as a result of its inability to penetrate the bacterial cell wall. Temporarily masking the phosphonate moiety as a prodrug has the potential to solve both issues. We report the application of two amino acid based prodrug approaches on a fosmidomycin surrogate. Conversion of the phosphonate moiety into tyrosine-derived esters increases the in vitro activity against asexual blood stages of P. falciparum, while phosphonodiamidate prodrugs display promising antitubercular activities. Selected prodrugs were tested in vivo in a P. berghei malaria mouse model. These results indicate good in vivo antiplasmodial potential.

Ethnopharmacological relevance: An aqueous decoction of root bark of Alstonia congensis Engl. (Apocynaceae) is used in several African countries to treat various ailments including malaria. Materials and methods: Extracts of different polarity and isolated constituents were tested in vitro for their antiplasmodial activity against the chloroquine-resistant strain Plasmodium falciparum K1 and the chloroquine-sensitive strain P. falciparum NF54A19A, as well as for their cytotoxic effects againt MRC-5 cells (human lung fibroblasts). Extracts and fractions were evaluated in vivo against the chloroquine-resistant strain P. yoelii N67 and the chloroquine-sensitive strain P. berghei berghei ANKA. Results: The aqueous extract, the 80% methanol extract and the alkaloid-enriched extract exhibited strong antiplasmodial activity against P. falciparum K1 with IC50 values < 10 mu g/ml and against P. falciparum NF54 A19A with IC50 values < 0.02 mu g/ml. In vivo against P. yoelii N67, at the highest oral dose of 400 mg/kg body weight, all extracts produced 70-73% chemosuppression, while against P. berghei berghei, more than 75% chemosuppression was observed. With regard to the isolated constituents, boonein was inactive in vitro against P. falci-parwn K-1 (IC50 > 64 mu M), while echitamine, 6,7-seco-angustilobine B and beta-amyrin exhibited moderate activity (IC50 < 30 mu M). Against P. falciparum NF54 A19A, boonein was inactive (IC50 > 64 mu M), while echitamine, 6,7-secoangustilobine and beta-amyrin showed moderate IC50 values of 11.07, 21.26 and 40.70 mu M, respectively. Conclusion: These results demonstrated that all extracts from A. congensis root bark possessed antiplasmodial activity in vitro and in vivo. They can be used as raw materials for the preparation of ameliorated remedies for the treatment of uncomplicated malaria. The observed antiplasmodial activity may be due in part to the presence of indole alkaloids.

A series of eleven double prodrug derivatives of a fosmidomycin surrogate were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. A pivaloyloxymethyl (POM) phosphonate prodrug modification was combined with various prodrug derivatisations of the hydroxamate moiety. The majority of compounds showed activity comparable with or inferior to fosmidomycin against P. falciparum. N-benzyl substituted carbamate prodrug 6f was the most active antimalarial analog with an IC50 value of 0.64 mu M. Contrary to fosmidomycin and parent POM-prodrug 5, 2-nitrofuran and 2-nitrothiophene prodrugs 6i and 6j displayed promising antitubercular activities.

The bioactive petroleum ether fraction of Verbesina encelioides, previously studied by the authors, was chosen for the isolation of antiprotozoal metabolites. Pseudotaraxasterol-3β-acetate (1), benzyl 2,6-dimethoxy benzoate (2), 16β-hydroxy-pseudotaraxasterol-3β-palmitate (3) and pseudotaraxasterol (4), in addition to β-sitosterol glucoside (5) and β-sitosterol galactoside (6) were isolated and identified based on one-dimensional and two-dimensional spectral analysis. This is the first report describing (3) and (6) in genus Verbesina. The isolated compounds were tested in vitro against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Cytotoxicity was evaluated on MRC-5 cells. Compound 1 showed moderate to weak activity against L. infantum T. brucei and P. falciparum and was inactive against T. cruzi. Compound 3 showed moderate activity against L. infantum, compound 4 revealed weak activity against T. cruzi, while 5 and 6 were inactive against all tested protozoa. All compounds were non-cytotoxic. The isolated constituents showed less antiprotozoal activity than the crude fraction.

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Tuberculosis (TB) still has a major impact on public health. In order to efficiently eradicate this life-threatening disease, the exploration of novel anti-TB drugs is of paramount importance. As part of our program to design new 2-azaanthraquinones with anti-mycobacterial activity, various “out-of-plane” tetrahydro- and octahydrobenzo[j]phenanthridinediones were synthesized. In this study, the scaffold of the most promising hits was further optimized in an attempt to improve the bioactivity and to decrease enzymatic degradation. The rudiment bio-evaluation of a small library of fluorinated tetrahydrobenzo[j]phenanthridine-7,12-dione derivatives indicated no significant improvement of the bio-activity against intracellular and extracellular Mycobacterium tuberculosis (Mtb). Though, the derivatives showed an acceptable toxicity against J774A.1 macrophages and early signs of genotoxicity were absent. All derivatives showed to be metabolic stabile in the presence of both phase I and phase II murine or human microsomes. Finally, the onset of reactive oxygen species within Mtb after exposure to the derivatives was measured by electron paramagnetic resonance (EPR). Results showed that the most promising fluorinated derivative is still a possible candidate for the subversive inhibition of mycothione reductase.

Prevention of severe lower respiratory tract infections in infants caused by the human respiratory syncytial virus (hRSV) remains a major public health priority. Currently, the major focus of vaccine development relies on the RSV fusion (F) protein since it is the main target protein for neutralizing antibodies induced by natural infection. The protein conserves 5 N-glycosylation sites, two of which are located in the F2 subunit (N27 and N70), one in the F1 subunit (N500) and two in the p27 peptide (N116 and N126). To study the influence of the loss of one or more N-glycosylation sites on RSV F immunogenicity, BALB/c mice were immunized with plasmids encoding RSV F glycomutants. In comparison with F WT DNA immunized mice, higher neutralizing titres were observed following immunization with F N116Q. Moreover, RSV A2-K-line19F challenge of mice that had been immunized with mutant F N116Q DNA was associated with lower RSV RNA levels compared with those in challenged WT F DNA immunized animals. Since p27 is assumed to be post-translationally released after cleavage and thus not present on the mature RSV F protein, it remains to be elucidated how deletion of this glycan can contribute to enhanced antibody responses and protection upon challenge. These findings provide new insights to improve the immunogenicity of RSV F in potential vaccine candidates.

Several methods have been developed for the detection of Leishmania, mostly targeting the minicircle kinetoplast DNA (kDNA). A new RNA real-time quantitative PCR (qPCR) assay was developed targeting the conserved and highly expressed spliced-leader (SL) mini-exon sequence. This study compared the limits of detection of various real-time PCR assays in hamsters infected with Leishmania infantum, in spiked human blood, and in clinical blood samples from visceral leishmaniasis patients. The SL-RNA assay showed an excellent analytical sensitivity in tissues (0.005 and 0.002 parasites per mg liver and spleen, respectively) and was not prone to false-positive reactions. Evaluation of the SL-RNA assay on clinical samples demonstrated lower threshold cycle values than the kDNA qPCR, an excellent interrun stability of 97%, a 93% agreement with the kDNA assay, and an estimated sensitivity, specificity, and accuracy of 93.2%, 94.3%, and 93.8%, respectively. The SL-RNA qPCR assay was equally efficient for detecting Leishmania major, Leishmania tropica, Leishmania mexicana, Leishmania guayensis, Leishmania panamensis, Leishmania braziliensis, L. infantum, and Leishmania donovani and revealed similar SL-RNA levels in the different species and the occurrence of polycistronic SL-containing transcripts in Viannia species. Collectively, this single SL-RNA qPCR assay enables universal Leishmania detection and represents a particularly useful addition to the widely used kDNA assay in clinical studies in which the detection of viable parasites is pivotal to assess parasitological cure.

Chemotherapy is currently the only effective approach to treat all forms of leishmaniasis. However, its effectiveness is severely limited due to high toxicity, long treatment length, drug resistance, or inadequate mode of administration. As a consequence, there is a need to identify new molecular scaffolds and targets as potential therapeutics for the treatment of this disease. We report a small series of 1,2‐substituted‐1H‐benzo[d]imidazole derivatives (9ad) showing affinity in the submicromolar range (Ki = 0.150.69 μM) toward Leishmania mexicanaCPB2.8ΔCTE, one of the more promising targets for antileishmanial drug design. The compounds confirmed activity in vitro against intracellular amastigotes of Leishmania infantum with the best result being obtained with derivative 9d (IC50 = 6.8 μM), although with some degree of cytotoxicity (CC50 = 8.0 μM on PMM and CC50 = 32.0 μM on MCR‐5). In silico molecular docking studies and ADME‐Tox properties prediction were performed to validate the hypothesis of the interaction with the intended target and to assess the drug‐likeness of these derivatives.

In an effort to identify novel molecular warheads able to inhibit Leishmania mexicana cysteine protease CPB2.8CTE, fused benzo[b]thiophenes and ,'-triketones emerged as covalent inhibitors binding the active site cysteine residue. Enzymatic screening showed a moderate-to-excellent activity (12%-90% inhibition of the target enzyme at 20m). The most promising compounds were selected for further profiling including in vitro cell-based assays and docking studies. Computational data suggest that benzo[b]thiophenes act immediately as non-covalent inhibitors and then as irreversible covalent inhibitors, whereas a reversible covalent mechanism emerged for the 1,3,3'-triketones with a Y-topology. Based on the predicted physicochemical and ADME-Tox properties, compound 2b has been identified as a new drug-like, non-mutagen, non-carcinogen, and non-neurotoxic lead candidate.

Objectives Widespread antimony resistance in the Indian subcontinent has enforced a therapy shift in visceral leishmaniasis treatment primarily towards miltefosine and secondarily also towards paromomycin. In vitro selection of miltefosine resistance in Leishmania donovani turned out to be quite challenging. Although no increase in IC50 was detected in the standard intracellular amastigote susceptibility assay, promastigote back-transformation remained positive at high miltefosine concentrations, suggesting a more resistant phenotype. This observation was explored in a large set of Nepalese clinical isolates from miltefosine cure and relapse patients to assess its predictive value for patient treatment outcome. Methods The predictive value of the promastigote back-transformation for treatment outcome of a set of Nepalese L. donovani field isolates (n = 17) derived from miltefosine cure and relapse patients was compared with the standard susceptibility assays on promastigotes and intracellular amastigotes. Results In-depth phenotypic analysis of the clinical isolates revealed no correlation between the different susceptibility assays, nor any clear link to the actual treatment outcome. In addition, the clinical isolates proved to be phenotypically heterogeneous, as reflected by the large variation in drug susceptibility among the established clones. Conclusions This in vitro laboratory study shows that miltefosine treatment outcome is not necessarily exclusively linked with the susceptibility profile of pre-treatment isolates, as determined in standard susceptibility assays. The true nature of miltefosine treatment failures still remains ill defined.

Fosmidomycin inhibits IspC (Dxr, 1-deoxy-D-xylulose 5-phosphate reductoisomerase), a key enzyme in nonmevalonate isoprenoid biosynthesis that is essential in Plasmodium falciparum. The drug has been used successfully to treat malaria patients in clinical studies, thus validating IspC as an antimalarial target. However, improvement of the drugs pharmacodynamics and pharmacokinetics is desirable. Here, we show that the conversion of the phosphonate moiety into acyloxymethyl and alkoxycarbonyloxymethyl groups can increase the in vitro activity against asexual blood stages of P. falciparum by more than 1 order of magnitude. We also synthesized double prodrugs by additional esterification of the hydroxamate moiety. Prodrugs with modified hydroxamate moieties are subject to bioactivation in vitro. All prodrugs demonstrated improved antiplasmodial in vitro activity. Selected prodrugs and parent compounds were also tested for their cytotoxicity toward HeLa cells and in vivo in a Plasmodium berghei malaria model as well as in the SCID mouse P. falciparum model.

Human African trypanosomiasis (HAT), also known as sleeping sickness is a parasitic disease transmitted by the bite of the 'Glossina' insect, commonly known as the tsetse fly. This disease affects mostly poor populations living in remote rural areas of Africa. Untreated, it is usually fatal. Currently, safe and effective treatments against this disease are lacking. Phenotypic screening of triazine non-nucleoside HIV-1 reverse transcriptase inhibitors (monomers) resulted in potent and selective antitrypanosomal compounds. This serendipitous discovery and the presence of dimers in many compounds active against these neglected tropical diseases prompted us to investigate antitrypanosomal activity of triazine dimers. Optimization of the triazine dimers resulted in 3,3'-(((ethane-1,2-diylbis(azanediyl))bis(4-(mesityloxy)-1,3,5-triazine-6,2-diyl))bis(azanediyl))dibenzonitrile (compound 38), a compound with very potent in vitro and moderate in vivo antitrypanosomal activity.

Graphical abstract, A 900 compound nitroimidazole-based library derived from our pretomanid backup program with TB Alliance was screened for utility against human African trypanosomiasis (HAT) by the Drugs for Neglected Diseases initiative. Potent hits included 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides, which surprisingly displayed good metabolic stability and excellent cell permeability. Following comprehensive mouse pharmacokinetic assessments on four hits and determination of the most active chiral form, a thiazine oxide counterpart of pretomanid (24) was identified as the best lead. With once daily oral dosing, this compound delivered complete cures in an acute infection mouse model of HAT and increased survival times in a stage 2 model, implying the need for more prolonged CNS exposure. In preliminary SAR findings, antitrypanosomal activity was reduced by removal of the benzylic methylene but enhanced through a phenylpyridine-based side chain, providing important direction for future studies.