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Advanced nanoscale antimicrobials, originated from the combination of noble metal nanoparticles (NPs) with conventional antimi-crobial drugs, are considered the next generation of antimicrobial agents. Therefore, there is an increasing demand for rapid, eco-friendly, and relatively inexpensive synthetic approaches for the preparation of nontoxic metallic nanostructures endowed with unique physicochemical properties. Recently, we have proposed a straightforward synthetic strategy that exploits the properties of polymeric beta-cyclodextrin (PolyCD) to act as both the reducing and stabilizing agent to produce monodispersed and stable gold-based NPs either as monometallic (nanoG) structures or core-shell bimetallic (nanoGS) architectures with an external silver layer. Here, we describe the preparation of a supramolecular assembly between nanoGS and pentamidine, an antileishmanial drug endowed with a wide range of therapeutic properties (i.e., antimicrobial, anti-inflammatory, and anticancer). The physicochemical characterization of the supramolecular assembly (nanoGSP) in terms of size and colloidal stability was investigated by complemen-tary spectroscopic techniques, such as UV-vis, zeta-potential, and dynamic light scattering (DLS). Furthermore, the role of PolyCD during the reduction/stabilization of metal NPs was investigated for the first time by NMR spectroscopy.

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.

Current drugs for Chagas disease have long treatment regimens with occurrence of adverse drug effects leading to poor treatment compliance. Novel and efficacious medications are therefore highly needed. We previously reported on the discovery of NPD-0227 (2-isopropyl-5-(4-methoxy-3-(pyridin-3-yl)phenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one) as a potent in vitro inhibitor of Trypanosoma cruzi (pIC50 = 6.4) with 100-fold selectivity over human MRC-5 cells. The present work describes a SAR study on the exploration of substituents on the phenylpyrazolone nitrogen. Modifications were either done directly onto this pyrazolone nitrogen or alternatively by introducing a piperidine linker. Attention was pointed toward the selection of substituents with a cLogP preferably below NPD-0227s cLogP of 3.5. Generally the more apolar compounds showed better activities then molecules with cLogPs 50 = 6.4) and promising selectivities. While the potency could not be improved, valuable SAR was obtained. Furthermore the introduction of a piperidine linker offers new opportunities for derivatization as valuable novel starting points for future T. cruzi drug discovery.

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.

Schistosomiasis is a neglected tropical disease with high morbidity. Recently, the Schistosoma mansoni phosphodiesterase SmPDE4A was suggested as a putative new drug target. To support SmPDE4A targeted drug discovery, we cloned, isolated, and biochemically characterized the full-length and catalytic domains of SmPDE4A. The enzymatically active catalytic domain was crystallized in the apo-form (PDB code: 6FG5) and in the cAMP- and AMP-bound states (PDB code: 6EZU). The SmPDE4A catalytic domain resembles human PDE4 more than parasite PDEs because it lacks the parasite PDE-specific P-pocket. Purified SmPDE4A proteins (full-length and catalytic domain) were used to profile an in-house library of PDE inhibitors (PDE4NPD toolbox). This screening identified tetrahydrophthalazinones and benzamides as potential hits. The PDE inhibitor NPD-0001 was the most active tetrahydrophthalazinone, whereas the approved human PDE4 inhibitors roflumilast and piclamilast were the most potent benzamides. As a follow-up, 83 benzamide analogs were prepared, but the inhibitory potency of the initial hits was not improved. Finally, NPD-0001 and roflumilast were evaluated in an in vitro anti-S. mansoni assay. Unfortunately, both SmPDE4A inhibitors were not effective in worm killing and only weakly affected the egg-laying at high micromolar concentrations. Consequently, the results with these SmPDE4A inhibitors strongly suggest that SmPDE4A is not a suitable target for anti-schistosomiasis therapy.

Мета . Надати скорочений огляд літературних даних стосовно будови коронавірусу людини SARS-CoV-2, механізму його репродукції та ролі вірусних протеаз у цьому процесі. За допомогою інструментів комп’ютерного моделювання проаналізувати здатність відомих противірусних агентів та de novo синтезованих сполук зв’язувати та пригнічувати головну протеазу коронавірусу. Результати та їх обговорення . Коронавірусна хвороба COVID-19 за останні місяці стала викликом для всього світу. З огляду на швидке поширення та важкий перебіг COVID-19 у значної частини населення існує нагальна потреба в ефективних лікарських засобах і відповідних протоколах лікування, які, на жаль, поки що відсутні. На теперішній час пошук молекул з прийнятним профілем токсичності, здатних пригнічувати та/або припиняти репродукцію коронавірусу SARS-CoV-2 в організмі людини, є вельми актуальним. У представленій роботі на основі даних про будову головної протеази M pro SARS-CoV-2 було проведено віртуальний скринінг та молекулярний докінг як відомих противірусних агентів, так і нових синтезованих сполук. Виявлені в ході дослідження закономірності можуть бути корисними для пошуку та розробки нових противірусних лікарських засобів для боротьби з захворюванням COVID-19 та іншими коронавірусними інфекційними хворобами. Аналіз результатів розрахунків фізико-хімічних характеристик противірусних агентів та визначення сайтів їх зв’язування з головною вірусною протеазою M pro дозволяє дати оптимістичну оцінку можливості створення нових лікарських засобів на основі відомих противірусних лікарських засобів або їх модифікованих аналогів. Експериментальна частина . На основі аналізу новітніх досліджень кристалічної структури головної протеази вірусу M pro у комплексі з різними інгібіторами (Protein Data Bank http://www.rcsb.org/pdb , код структури – 6LU7) проведено віртуальний скринінг та молекулярний докінг 100 відомих противірусних агентів та 50 нових синтезованих сполук. Наведено дані скринінгу in vitro антималярійної активності синтезованих сполук. Для всіх потенційних інгібіторів головної протеази вірусу було розраховано параметри зв’язування та фізико-хімічні параметри взаємодії «ліганд–білок»: показник спорідненості (BAS), енергію зв’язування, ліпофільність (clogP) та топологічний індекс площі поверхні полярних груп молекули ліганду (TPSA). Вивчення структури білка та ліганду проводили за допомогою графічних програмних пакетів Jmol, PyMol та Avogadro. Віртуальний скринінг та молекулярний докінг, а також аналіз отриманих результатів проводили за допомогою програмного комплексу LigandScout 4.4. Дані щодо антималярійної активності 50 синтезованих сполук були отримані в Лабораторії мікробіології, паразитології та гігієни Антверпенського університету (Бельгія). Висновки . За результатами віртуального скринінгу та молекулярного докінгу з білком 6LU7 було встановлено, що низка відомих противірусних лікарських засобів має певну перспективу використання як інгібіторів головної протеази коронавірусу SARS-CoV-2, а такі субстанції, як ремдесивір та ритонавір характеризуються більш високою активністю, ніж референтна сполука з комплексу 6LU7. Молекулярний докінг ряду нових, нещодавно синтезованих сполук з доведеною in vitro антималярійною активністю показав, що сполуки L1 – L6 можуть стати перспективними кандидатами для подальшої модифікації та розробки нових противірусних лікарських засобів для боротьби з коронавірусною інфекцією. Received : 02.04.2020 Revised : 23.05.2020 Accepted : 29.05.2020

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.

Leishmaniasis is a neglected parasitic disease caused by over 20 different Leishmania species. Current treatments often rely on harsh regimes of pentavalent antimonials such as sodium stibogluconate, while more recent drugs suffer other shortcomings such as low stability and rapid emergence of treatment failure, amongst others. Furthermore, the effectiveness of drugs varies depending on the infecting Leishmania species, thus there is an urgent need for new and effective anti-leishmanial drugs. Screening of an in-house compound library identified the hexahydrophthalazinone NPD-2942 as a low micromolar hit with a pIC50 of 5.8 against L. infantum and a pIC50 of 4.6 for cytotoxicity against human MRC-5 fibroblasts. To derive structure–activity relationships, we modified the cyclohexyl ring of the hexahydrophthalazinone scaffold and 1,2,3-triazoles were attempted as replacement for the pyrazole ring, amongst others. Ultimately, the 2,3-pyrazole-substituted hexahydrophthalazinone NPD-1289 was identified as the most potent analogue in this series with a pIC50 of 6.3, although some cytotoxicity toward MRC-5 cells (pIC50=5.1) was recorded as well. Replacement of the unsubstituted 2,3-pyrazole with 1,2,3-triazoles led to compounds with lower anti-leishmanial activity. The current scaffold is a valuable new starting point for optimization toward novel anti-leishmanial drugs.

13 p.-7 fig.-5 tab.-1 graph. abst., A previous phenotypic screening campaign led to the identification of a quinazoline derivative with promising in vitro activity against Schistosoma mansoni. Follow-up studies of the antischistosomal potential of this candidate are presented here. The in vivo studies in a S. mansoni mouse model show a significant reduction of total worms and a complete disappearance of immature eggs when administered concomitantly with praziquantel in comparison with the administration of praziquantel alone. This fact is of utmost importance because eggs are responsible for the pathology and transmission of the disease. Subsequently, the chemical optimisation of the structure in order to improve the metabolic stability of the parent compound was carried out leading to derivatives with improved drug-like properties. Additionally, the putative target of this new class of antischistosomal compounds was envisaged by using computational tools and the binding mode to the target enzyme, aldose reductase, was proposed., This study received funding from the EC 7th Framework Programme (FP7-HEALTH-2013-INNOVATION-1, PDE4NPD no.602666), RICET (RD16/0027/0010), FEDER funds and MECD [Grant FPU15/1465 to V. S.-P.].

Animal trypanosomiasis (AT) is a parasitic disease with high socio-economic impact. Given the limited therapeutic options and problems of toxicity and drug resistance, this study assessed redirecting our previously identified antitrypanosomal nucleosides for the treatment of AT. Promising hits were identified with excellent in vitro activity across all important animal trypanosome species. Compound 7, an inosine analogue, and our previously described lead compound, 3???-deoxytubercidin (8), showed broad spectrum anti-AT activity, metabolic stability in the target host species and absence of toxicity, but with variable efficacy ranging from limited activity to full cure in mouse models of Trypanosoma congolense and T. vivax infection. Several compounds show promise against T. evansi (surra) and T. equiperdum (dourine). Given the preferred target product profile for a broadspectrum compound against AT, this study emphasizes the need to include T. vivax in the screening cascade given its divergent susceptibility profile and provides a basis for lead optimization towards such broad spectrum anti-AT compound.

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.

Background The protozoan Trypanosoma cruzi is auxotrophic for purines and causes Chagas’ disease (CD), a neglected illness affecting >6 million people. Combining the 3-deoxyribofuranose part of cordycepin with the modified purine ring of a nucleoside ‘hit’ led to the discovery of 4-amino-5-(4-chlorophenyl)-N7-(3′-deoxy-β-d-ribofuranosyl)-pyrrolo[2,3-d]pyrimidine (Cpd1), revealing promising anti-T. cruzi activity. Objectives To further evaluate Cpd1 in vitro and in vivo to fully assess its therapeutic potential against CD, covering cell culture sterilization through washout assays, drug combination with benznidazole and long-term administration in T. cruzi-infected mice. Results Although less susceptible to Cpd1 than amastigotes, trypomastigotes present an impaired capacity to successfully establish intracellular infection of cardiac cultures. Combination of benznidazole with Cpd1 indicated no interaction (additive effect) (FIC index = 0.72) while administration to mice at one-tenth of the optimal dose (2.5 mg/kg and 10 mg/kg for Cpd1 and benznidazole, respectively) suppressed parasitaemia but failed to avoid mortality. Long-term treatment (60 days) gave a rapid drop of the parasitaemia (>98% decline) and 100% mice survival but only 16% cure. In vitro washout experiments demonstrated that although parasite release into the supernatant of infected cardiac cultures was reduced by >94%, parasite recrudescence did occur after treatment. Conclusions Parasite recrudescence did occur after treatment corroborating the hypothesis of therapeutic failure due to subpopulations of dormant forms and/or genetic factors in persister parasites involved in natural drug resistance.

Trypanosoma cruzi is the causative pathogen of Chagas disease and the main culprit for cardiac-related mortality in Latin-America triggered by an infective agent. Uncapable of synthesizing purines de novo, this parasite depends on acquisition and processing of host-derived purines, making purine (nucleoside) analogues a potential source of antitrypanosomal agents. In this respect, hitherto 7-deazaadenosine (tubercidin) analogues attracted most attention. Here, we investigated analogues with an additional nitrogen (N1) removed. Structure-activity relationship investigation showed that C7 modification afforded analogues with potent antitrypanosomal activity. Halogens and small, linear carbon-based substituents were preferred. Compound 11 proved most potent in vitro, showed full suppression of parasitemia in a mouse model of acute infection and elicited 100 % animal survival after oral dosing at 25 mg/kg b.i.d. for five and fifteen days. Cyclophosphamide-induced immunosuppression led to recrudescence. Washout experiments demonstrated a lack of complete clearance of infected cell cultures, potentially explaining the in vivo results.

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

As over 6 million people are infected with Chagas disease and only limited therapeutic options are available, there is an urgent need for novel drugs. The involvement of cyclic nucleotide phosphodiesterases (PDE) in the lifecycle and biological fitness of a number of protozoan parasites has been described and several of these enzymes are thought to be viable drug targets. Within this context, a PDE-focused library was screened for its ability to affect the viability of Trypanosoma cruzi parasites. 5-(3-(Benzyloxy)-4-methoxyphenyl)-2-isopropyl-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (4), previously reported as a human PDE4 inhibitor, was identified as a hit. Upon optimization on three positions of the phenylpyrazolone scaffold, 2-isopropyl-5-(4-methoxy-3-(pyridin-3-yl)phenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (34) proved to be the most active compound against intracellular forms of T. cruzi (pIC 50 = 6.4) with a 100-fold selectivity with respect to toxicity toward human MRC-5 cells. Evaluation on different life stages and clinically relevant T. cruzi strains revealed that the phenylpyrazolones are not active against the bloodstream form of the Y strain but show submicromolar activity against the intracellular form of the Y- and Tulahuen strains as well as against the nitro-drug-resistant Colombiana strain. In vitro screening of phenylpyrazolones against TcrPDEB1, TcrPDEC, and TcrCYP51 showed that there was a poor correlation between enzyme inhibition and the observed phenotypic effect. Among the most potent compounds, both TcrCYP51 and non-TcrCYP51 inhibitors are identified, which were both equally able to inhibit T. cruzi in vitro.

Leishmaniasis causes high mortality and morbidity in tropical and subtropical regions of Africa, Asia, the Americas and southern Europe, and is characterized by diverse clinical manifestations. As a neglected tropical disease, limited resources are allocated for antileishmanial drug discovery. The Leishmania parasite is deficient in de novo purine synthesis, and therefore acquires purines from the host and processes these using a purine salvage pathway. By making use of purine transport systems and interfering with this salvage pathway, purine (nucleoside) analogues might exert a selective detrimental impact on its growth and survival. In vitro screening of an in-house purine nucleoside library and analogue synthesis afforded the 6-methyl-7-(2-pyridyl)-7-deazapurine ribonucleoside analogue 18 as a promising hit. Optimization of the 7-substituent afforded 31 and 32 which displayed potent activity against wild-type and resistant L. infantum, intracellular amastigote and extracellular promastigote forms, and favorable selectivity versus primary mouse macrophages (M phi) and MRC-5 cells. Encouraged by the favorable in vitro metabolic stability of 32, an in vivo study was performed using an early curative L. infantum hamster model. When orally administrated at 50 mg/kg once daily (s.i.d) for 10 days, 32 was devoid of side effects, however, it only poorly reduced amastigote burdens in the major target organs.

Chagas disease is a tropical infectious disease resulting in progressive organ-damage and currently lacks efficient treatment and vaccine options. The causative pathogen, Trypanosoma cruzi, requires uptake and processing of preformed purines from the host because it cannot synthesize these de novo, instigating the evaluation of modified purine nucleosides as potential trypanocides. By modifying the pyrimidine part of a previously identified 7-aryl-7-deazapurine nucleoside, we found that substitution of a 6-methyl for a 6-amino group allows retaining T. cruzi amastigote growth inhibitory activity but confers improved selectivity towards mammalian cells. By keeping the 6-methyl group unaltered, and introducing different 7-aryl groups, we identified several analogues with sub-micromolar antitrypanosomal activity. The 7-(4-chlorophenyl) analogue 14, which was stable in microsomes, was evaluated in an acute mouse model. Oral administration of 25 mg/kg b.i.d. suppressed peak parasitemia and protected mice from infection-related mortality, gave similar reductions as the reference drug of blood parasite loads determined by qPCR, but as benznidazole failed to induce sterile cure in the short time period of drug exposure (5 days).

Several members of the 3′,5′-cyclic nucleotide phosphodiesterase (PDE) family play an essential role in cellular processes, which has labeled them as interesting targets for various diseases. The parasitic protozoan Trypanosoma brucei, causative agent of human African trypanosomiasis, contains several cyclic AMP specific PDEs from which TbrPDEB1 is validated as a drug target. The recent discovery of selective TbrPDEB1 inhibitors has increased their potential for a novel treatment for this disease. Compounds characterized by a rigid biphenyl tetrahydrophthalazinone core structure were used as starting point for the exploration of novel TbrPDEB1 inhibitors. Using a virtual screening campaign and structure-guided design, diaryl ether substituted phthalazinones were identified as novel TbrPDEB1 inhibitors with IC50 values around 1 μM against T. brucei. This study provides important structure-activity relationship (SAR) information for the future design of effective parasite-specific PDE inhibitors.

[Image: see text] Antitubercular 7-substituted 2-nitroimidazo[2,1-b][1,3]oxazines were previously shown to exhibit potent antileishmanial and antitrypanosomal activities, culminating in a new clinical investigational drug for visceral leishmaniasis (DNDI-0690). To offset development risks, we continued to seek further leads with divergent candidate profiles, especially analogues possessing greater aqueous solubility. Starting from an efficacious monoaryl derivative, replacement of the side chain ether linkage by novel amine, amide, and urea functionality was first explored; the former substitution was well-tolerated in vitro and in vivo but elicited marginal alterations to solubility (except through a less stable benzylamine), whereas the latter groups resulted in significant solubility improvements (up to 53-fold) but an antileishmanial potency reduction of at least 10-fold. Ultimately, we discovered that O-carbamate 66 offered a more optimal balance of increased solubility, suitable metabolic stability, excellent oral bioavailability (100%), and strong in vivo efficacy in a visceral leishmaniasis mouse model (97% parasite load reduction at 25 mg/kg).

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.

The kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are the causative agents of neglected tropical diseases with a serious burden in several parts of the world. These parasites are incapable of synthesizing purines de novo, and therefore rely on ingenious purine salvage pathways to acquire and process purines from their host. Purine nucleoside analogs that may interfere with these pathways therefore constitute a privileged source of new antikinetoplastid agents. In this study, we synthetized a collection of C-nucleosides employing five different heterocyclic nucleobase surrogates. C-nucleosides are chemically and enzymatically stable and allow for extensive structural modification. Inspired by earlier 7-deazaadenosine nucleosides and known antileishmanial C-nucleosides, we introduced different modifications tailored towards antikinetoplastid activity. Both adenosine and inosine analogs were synthesized with the aim of discovering new antikinetoplastid hits and expanding knowledge of structure-activity relationships. Several promising hits with potent activity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum were discovered, and the nature of the nucleobase surrogate was found to have a profound influence on the selectivity profile of the compounds. (C) 2020 Elsevier Masson SAS. All rights reserved.

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.

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 de novo purine synthesis, they depend on purine salvage pathways that allow them to acquire and process purines from the host to meet their demands. Purine nucleoside analogues therefore constitute a logical source of potential antiparasitic agents. Earlier optimization efforts of the natural product tubercidin (7-deazaadenosine) involving modifications to the nucleobase 7-position and the ribofuranose 3'-position led to analogues with potent anti-Trypanosoma brucei and anti-Trypanosoma cruzi activities. In this work, we report the design and synthesis of pyrazolo[3,4-d]pyrimidine nucleosides with 3'- and 7-modifications and assess their potential as anti-Trypanosoma cruzi and antileishmanial agents. One compound was selected for in vivo evaluation in an acute Chagas disease mouse model.

Chagas disease and leishmaniasis are two poverty-related neglected tropical diseases that cause high mortality and morbidity. Current treatments suffer from severe limitations and novel, safer and more effective drugs are urgently needed. Both Trypanosoma cruzi and Leishmania are auxotrophic for purines and absolutely depend on uptake and assimilation of host purines. This led us to successfully explore purine nucleoside analogues as chemotherapeutic agents against these and other kinetoplastid infections. This study extensively explored the modification of the 6-amino group of tubercidin, a natural product with trypanocidal activity but unacceptable toxicity for clinical use. We found that mono-substitution of the amine with short alkyls elicits potent and selective antitrypanosomal and antileishmanial activity. The methyl analogue 15 displayed the best in vitro activity against both T. cruzi and L. infantum and high selectivity versus host cells. Oral administration for five consecutive days in an acute Chagas disease mouse model resulted in significantly reduced peak parasitemia levels (75, 89 and 96% with 12.5, 25 and 50 mg/kg/day, respectively). as well as increased animal survival rates with the lower doses (83 and 67% for 12.5 and 25 mg/kg/day, respectively).

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.

Current drugs for Chagas disease have long treatment regimens with occurrence of adverse drug effects leading to poor treatment compliance. Novel and efficacious medications are therefore highly needed. We previously reported on the discovery of NPD-0227 (2-isopropyl-5-(4-methoxy-3-(pyridin-3-yl)phenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one) as a potent in vitro inhibitor of Trypanosoma cruzi (pIC50 = 6.4) with 100-fold selectivity over human MRC-5 cells. The present work describes a SAR study on the exploration of substituents on the phenylpyrazolone nitrogen. Modifications were either done directly onto this pyrazolone nitrogen or alternatively by introducing a piperidine linker. Attention was pointed toward the selection of substituents with a cLogP preferably below NPD-0227s cLogP of 3.5. Generally the more apolar compounds showed better activities then molecules with cLogPs

Pyrazolones are heterocyclic compounds with interesting biological properties. Some derivatives inhibit phosphodiesterases (PDEs) and thereby increase the cellular concentration of cyclic AMP (cAMP), which plays a vital role in the control of metabolism in eukaryotic cells, including the protozoan Trypanosoma cruzi , the etiological agent of Chagas disease (CD), a major neglected tropical disease. In vitro phenotypic screening identified a 4-bromophenyl-dihydropyrazole dimer as an anti- T. cruzi hit and 17 novel pyrazolone analogues with variations on the phenyl ring were investigated in a panel of phenotypic laboratory models. Potent activity against the intracellular forms (Tulahuen and Y strains) was obtained with 50% effective concentration (EC 50 ) values within the 0.17 to 3.3 μM range. Although most were not active against bloodstream trypomastigotes, an altered morphology and loss of infectivity were observed. Pretreatment of the mammalian host cells with pyrazolones did not interfere with infection and proliferation, showing that the drug activity was not the result of changes to host cell metabolism. The pyrazolone NPD-227 increased the intracellular cAMP levels and was able to sterilize T. cruzi -infected cell cultures. Thus, due to its high potency and selectivity in vitro , and its additive interaction with benznidazole (Bz), NPD-227 was next assessed in the acute mouse model. Oral dosing for 5 days of NPD-227 at 10 mg/kg + Bz at 10 mg/kg not only reduced parasitemia (>87%) but also protected against mortality (>83% survival), hence demonstrating superiority to the monotherapy schemes. These data support these pyrazolone molecules as potential novel therapeutic alternatives for Chagas disease.

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.

BackgroundIn eco-epidemiological studies,Leishmaniadetection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-LeishmaniaSYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection ofLeishmaniain field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts.MethodsField-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target forLeishmaniadetection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve.ResultsThe qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10−3and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.ConclusionsThis study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification ofLeishmaniain sand flies. The assay is inexpensive, sensitive and pan-Leishmaniaspecific, and accordingly an excellent assay for high-throughput screening in entomological research.

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.

BackgroundIn eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR Green quantitative PCR (qPCR) assay which specifically detects the conserved spliced-leader RNA (SL-RNA) sequence has recently been developed. This study comparatively assessed the SL-RNA assay performance for the detection of Leishmania in field and laboratory infected sand flies and in tissue samples from hyraxes as reservoir hosts.Principal findingsThe qPCRs targeting SL-RNA and kDNA performed equally well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL-RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL-RNA levels were approximately 3-fold lower in sand fly promastigotes (ΔCt 1.7). The theoretical limit of detection and quantification of the SL-RNA qPCR respectively reached down to 10−3 and 10 parasite equivalents. SL-RNA detection in stored hyrax samples was less efficient with some false negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.ConclusionThis study shows that a crude extraction method in combination with the SL-RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay provides complementary information to the standard kDNA assays, since it is pan-Leishmania specific and detects viable parasites, a prerequisite for identification of vectors and reservoirs.Author summaryIn order to identify vectors and reservoirs of Leishmania, a large number of sand fly and animal tissue samples needs to be screened, because the infection prevalence is generally low. Hence, sensitive low-cost methods are required for nucleic acid isolation and Leishmania detection. Most approaches amplify DNA targets, in particular minicircle kinetoplast DNA (kDNA). Recently, a qPCR was developed that detects the spliced-leader RNA (SL-RNA) sequence, which is conserved among various Leishmania species and allows detection of viable parasites. We show that the SL-RNA qPCR is highly compatible with a low-cost, crude extraction approach and performs equally well on laboratory and field infected sand fly samples as kDNA qPCR assays. The assay can detect 10−3 parasite equivalent in sand flies and enables Leishmania quantification down to 10 parasites. We found that the copy number of SL-RNA is 3-fold lower in sand fly derived promastigotes compared to cultured promastigotes. SL-RNA detection in hyrax tissue samples appeared less efficient, which is presumably due to long-term storage without RNA stabilizing reagents. Overall, our assay is complementary to kDNA assays as it can identify viable Leishmania stages, which provides pivotal information for identification of reservoirs and vectors and their transmission capacity.

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.

A series of polymeric silver(I) complexes with Schiff base ligands containing pyridine and 3,5-halogen substituted phenol moieties were synthesized and characterized by spectroscopic methods and in the case of Ag6 also by X-ray crystallography. All silver(I) complexes (Ag1-Ag8) were evaluated for their biological activity against a panel of pathogens including the protozoa Trypanosoma cruzi, T. brucei, T. rhodesiense and Leishmania infantum; the bacteria Staphylococcus aureus, Escherichia coli, Mycobacterium tuberculosis H37Ra and the yeast Candida albicans. Cytotoxicity evaluation was carried out on human lung fibroblasts (MRC-5) and on primary peritoneal mouse macrophages. The most relevant result reveals antileishmanial activity potential with all complexes demonstrating higher potency than the reference drug miltefosine. Complexes with the best antiprotozoal profile (i.e., Ag2 and Ag7) were selected for incorporation into poly (lactic acid) nanoparticles (PLA NPs) with the aim to enhance selectivity. PLA/Ag2 NPs and PLA/Ag7 NPs exhibited adequate physicochemical properties, that is, average size of 263 +/- 60 nm and 225 +/- 6 nm, respectively, good entrapment efficiency (69% and 63%), loading content (6.2% and 5.7%) and stability. The cytotoxicity of PLA/Ag2 NPs and PLA/Ag7 NPs on MRC-5 cells was reduced with respect to the "free" metal complexes by similar to 2-fold and similar to 6-fold, respectively.

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.

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.

This article represents data regarding a study published in Toxicology in vitro entitled "in vitro CYP-mediated drug metabolism in the zebrafish (embryo) using human reference compounds" (Saad et al., 2017) [1]. Data were acquired with ultra-performance liquid chromatography - accurate mass mass spectrometry (UPLC-amMS). A full spectrum scan was conducted for the testosterone (TST) metabolites from the microsomal stability assay in zebrafish and humans. The microsomal proteins were extracted from adult zebrafish male (MLM) and female (FLM) livers, whole body homogenates of 96 h post fertilization larvae (EM) and a pool of human liver microsomes from 50 donors (HLM). Data are expressed as the abundance from the extracted ion chromatogram of the metabolites. (C) 2017 The Authors. Published by Elsevier Inc.

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.

As part of a quest for backups to the antitubercular drug pretomanid (PA-824), we investigated the unexplored 6-nitro-2,3-dihydroimidazo[2,1-b][1,3]-thiazoles and related-oxazoles. The nitroimidazothiazoles were prepared in high yield from 2-bromo-4-nitroimidazole via heating with substituted thiiranes and diisopropylethylamine. Equivalent examples of these two structural classes provided broadly comparable MICs, with 2-methyl substitution and extended aryloxymethyl side chains preferred; albeit, S-oxidised thiazoles were ineffective for tuberculosis. Favourable microsomal stability data for a biaryl thiazole (45) led to its assessment in an acute Mycobacterium tuberculosis mouse model, alongside the corresponding oxazole (48), but the latter proved to be more efficacious. In vitro screening against kinetoplastid diseases revealed that nitroimidazothiazoles were inactive versus leishmaniasis but showed interesting activity, superior to that of the nitroimidazooxazoles, against Chagas disease. Overall, "thio-delamanid" (49) is regarded as the best lead. (C) 2017 Elsevier Ltd. All rights reserved.

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.