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Objectives: Former studies demonstrated quick selection of paromomycin resistance for Leishmania infantum and Leishmania donovani accompanied by increased fitness. The present study aimed to interpret these findings in an epidemiological context by comparing infection of WT and experimentally derived paromomycin-resistant strains in the sand fly vector.Methods: Depending on the Leishmania species, Lutzomyia longipalpis and Phlebotomus perniciosus or Phlebotomus argentipes sand flies were artificially infected with procyclic promastigotes of WT and paromomycin-resistant L. infantum (MHOM/FR/96/LEM3323-cl4) or L. donovani (MHOM/NP/03/BPK275/0-cl18). The infection rate and gut/stomodeal valve colonization were determined to monitor parasite phenotypic behaviour within the vector. The impact of the previously described gain of fitness in the vertebrate host on infectivity for the vector was assessed by feeding L. longipalpis on Syrian golden hamsters heavily infected with either WT or paromomycin-resistant parasites.Results: WT and paromomycin-resistant Leishmania of both species behaved similarly in terms of infection and parasite location within the studied sand fly species. Blood feeding on infected hamsters did not reveal differences in acquisition of WT and paromomycin-resistant parasites, despite the higher organ burdens observed for the paromomycin-resistant strain. Strains remained resistant after passage in the vector.Conclusions: Although paromomycin-resistant parasites show an increased parasite fitness in vitro and in laboratory rodents, the intrinsic infection potential of paromomycin-resistant parasites remains unaltered in the sand fly. Of importance is the fact that paromomycin-resistant Leishmania are able to complete development in the natural vectors and produce stomodeal infection with metacyclic forms, which clearly suggests their potential to spread and circulate in nature. [ABSTRACT FROM AUTHOR]

Abstract: The tsetse fly (Glossina spp.) is an obligate blood-sucking insect that transmits different human-pathogenic and livestock threathening trypanosome species in Africa. To obtain more insight in the tsetse salivary function, some general aspects of the tsetse fly saliva and its composition were studied. Direct pH and protein content measurements revealed a moderately alkaline (pH∼8.0) salivary environment with approximately 4.3μg soluble proteins per gland and a constant representation of the major saliva proteins throughout the blood-feeding cycle. Although major salivary genes are constitutively expressed, upregulation of salivary protein synthesis within 48h after the blood meal ensures complete protein replenishment from day 3 onwards. Screening of a non-normalised Glossina morsitans morsitans λgt11 salivary gland expression library with serum from a saliva-immunized rabbit identified three full-length cDNAs encoding for novel salivary proteins with yet unknown functions: a 8.3kDa glycine/glutamate-rich protein (G. morsitans morsitans salivary gland protein Gmmsgp1), a 12.0kDa proline-rich protein (Gmmsgp2), and a 97.4kDa protein composed of a metallophosphoesterase/5′nucleotidase region with a glutamate/aspartate/asparagines-rich region (Gmmsgp3). [Copyright &y& Elsevier]

Streptococcus pneumoniae, the most common cause of bacterial pneumonia, has developed a wide range of virulence factors to evade the immune system of which the polysaccharide capsule is the most important one. Formation of this capsule is dependent on the cps gene locus, but also involves other genes—like galU. The pyrophosphorylase encoded by galU plays a role in the UDP-glucose metabolism of prokaryotes and is required for the biosynthesis of capsular polysaccharides. In this paper, the effect of a galU mutation leading to a dysfunctional UDP-glucose pyrophosphorylase (UDPG:PP) on in vitro biofilm biomass, adherence to lung epithelial cells and macrophage phagocytosis is studied. Last, in vivo virulence using a Galleria mellonella model has been studied. We show that the mutation improves streptococcal adherence to epithelial cells and macrophage phagocytosis in vitro, while there is no definitive correlation on biofilm formation between parent and mutant strains. Moreover, in vivo virulence is attenuated for all mutated strains. Together, these results demonstrate that a galU mutation in S. pneumoniae influences host cell interactions in vitro and in vivo and can strongly influence the outcome of a streptococcal infection. As such, UDPG:PP is worth investigating further as a potential drug target. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication. [ABSTRACT FROM AUTHOR]

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

A series of novel pyrazolyl amide-chalcones conjugates was synthesized in five steps and evaluated against a range of medically important kinetoplastid parasites including Trypanosoma cruzi, Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense and Leishmania infantum. In addition, the series was also tested for in vitro cytotoxicity activity against human lung fibroblasts and primary mouse macrophages. Among all synthetised compounds, 9b was found to be the most active against T. b. brucei with an IC 50 value of 0.51 ± 0.06 μM. Against T. b. rhodesiense, 9n was found to be the most potent with an IC 50 value of 0.46 ± 0.07 μM. While against L. infantum, 9a was found to be most active with an IC 50 value of 7.16 ± 1.88 μM. Based on the results and SAR, further modifications will be carried out to increase potency., (© 2024. The Author(s).)

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

Visceral leishmaniasis (VL) is characterized by an uncontrolled infection of internal organs such as the spleen, liver and bone marrow (BM) and can be lethal when left untreated. No effective vaccination is currently available for humans. The importance of B cells in infection and VL protective immunity has been controversial, with both detrimental and protective effects described. VL infection was found in this study to increase not only all analyzed B cell subsets in the spleen but also the B cell progenitors in the BM. The enhanced B lymphopoiesis aligns with the clinical manifestation of polyclonal hypergammaglobulinemia and the occurrence of autoantibodies. In line with earlier reports, flow cytometric and microscopic examination identified parasite attachment to B cells of the BM and spleen without internalization, and transformation of promastigotes into amastigote morphotypes. The interaction appears independent of IgM expression and is associated with an increased detection of activated lysosomes. Furthermore, the extracellularly attached amastigotes could be efficiently transferred to infect macrophages. The observed interaction underscores the potentially crucial role of B cells during VL infection. Additionally, using immunization against a fluorescent heterologous antigen, it was shown that the infection does not impair immune memory, which is reassuring for vaccination campaigns in VL endemic areas., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The protozoan parasites Plasmodium, Leishmania, and Trypanosoma are transmitted by hematophagous insects and cause severe diseases in humans. These infections pose a global threat, particularly in low-resource settings, and are increasingly extending beyond the current endemic regions. Tropism of parasites is crucial for their development, and recent studies have revealed colonization of noncanonical tissues, aiding their survival and immune evasion. Despite receiving limited attention, cumulative evidence discloses the respiratory system as a significant interface for host-pathogen interactions, influencing the course of (co)infection and disease onset. Due to its pathophysiological and clinical implications, we emphasize that further research is needed to better understand the involvement of the respiratory system and its potential to improve prevention, diagnosis, treatment, and interruption of the chain of transmission., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Three types of modifications of antileishmanial pyrazole lead compounds 7 and 8 were conducted to expand understanding of the relationships between structural features and antileishmanial/antitrypanosomal activity: (1) the pyrazole core was retained or replaced by a 1,2,4-triazole ring; (2) various aryl moieties including 2-fluorophenyl, pyridin-3-yl and pyrazin-2-yl rings were attached at 3-position of the core azole; (3) either arylmethylamino or ureido substituents were introduced at 5-position of the azole core. The synthesis followed established routes starting with esters 9 or 15 and anhydride 21. The synthesized 3-arylpyrazoles and 3-aryl-1,2,4-triazoles had only very low antileishmanial activity. The 2-fluorophenyl-substituted pyrazole 18c revealed the highest antileishmanial activity of this series of compounds, but its IC 50 value (20 μM) still indicates low activity. However, low micromolar antitrypanosomal activity was detected for the pyridin-3-yl-substituted pyrazoles 12b (IC 50 =4.7 μM) and 14a (IC 50 =2.1 μM). Their IC 50 values are comparable with the IC 50 values of the reference compounds benznidazole and nifurtimox. Whereas only low unspecific cytotoxicity at the primary peritoneal mouse macrophages (PMM) was detected, considerable cytotoxicity at MRC-5 human fibroblast cells was found for both pyrazoles 12b an 14a. The activity of pyrazole 12b against T. cruzi is 4-fold higher than its unspecific MRC-5 cytotoxicity., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Accurate detection of viable Leishmania parasites is critical for evaluating visceral leishmaniasis (VL) treatment response at an early timepoint. We compared the decay of kinetoplast DNA (kDNA) and spliced-leader RNA (SL-RNA) in vitro, in vivo, and in a VL patient cohort. An optimized combination of blood preservation and nucleic acid extraction improved efficiency for both targets. SL-RNA degraded more rapidly during treatment than kDNA, and correlated better with microscopic examination. SL-RNA quantitative polymerase chain reaction emerges as a superior method for dynamic monitoring of viable Leishmania parasites. It enables individualized treatment monitoring for improved prognoses and has potential as an early surrogate endpoint in clinical trials., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Metronidazole (2-methyl-5-nitro-1 H -imidazole-1-ethanol, MNZ) is a well-known and widely used drug for its excellent activity against various anaerobic bacteria and protozoa. The purpose of this study is to elucidate the ability of MNZ to form metal complexes with Cu 2+ and Zn 2+ and to demonstrate that complexation increases its bioactivity profile against different pathogenic microorganisms. The interaction of MNZ with Cu 2+ and Zn 2+ was investigated in NaCl aqueous solution under different conditions of temperature (15, 25, and 37 °C) and ionic strength (0.15, 0.5, and 1 mol L -1 ) by potentiometric and spectrophotometric titrations. The obtained speciation models include two species for the Cu 2+ -containing system, namely, CuL and CuL 2 , and three species for the Zn 2+ -containing system, namely, ZnLH, ZnL, and ZnLOH. The formation constants of the species were calculated and their dependence on temperature and ionic strength evaluated. Comparison of the sequestering ability of MNZ under physiological conditions revealed a capacity toward Cu 2+ higher than that toward Zn 2+ . A simulation under the same conditions also showed a significant percentage of the Cu 2+ -MNZ species. The biological assessments highlighted that the complexation of MNZ with Cu 2+ has a relevant impact on the potency of the drug against two Trypanosoma spp. (i.e., T. b. brucei and T. b. rhodesiense ) and one gram-(-) bacterial species (i.e., Escherichia coli ). It is noteworthy that the increased potency upon complexation with Cu 2+ did not result in cytotoxicity against MRC-5 human fetal lung fibroblasts and primary peritoneal mouse macrophages., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The bioluminescent Leishmania infantum BALB/c mouse model was used to evaluate the parasiticidal drug action kinetics of the reference drugs miltefosine, paromomycin, sodium stibogluconate, and liposomal amphotericin B. Infected mice were treated for 5 days starting from 7 days post-infection, and parasite burdens were monitored over time via bioluminescence imaging (BLI). Using nonlinear regression analyses of the BLI signal, the parasite elimination half-life ( t 1/2 ) in the liver, bone marrow, and whole body was determined and compared for the different treatment regimens. Significant differences in parasiticidal kinetics were recorded. A single intravenous dose of 0.5 mg/kg liposomal amphotericin B was the fastest acting with a t 1/2 of less than 1 day. Intraperitoneal injection of paromomycin at 320 mg/kg for 5 days proved to be the slowest with a t 1/2 of about 5 days in the liver and 16 days in the bone marrow. To conclude, evaluation of the cidal kinetics of the different antileishmanial reference drugs revealed striking differences in their parasite elimination half-lives. This BLI approach also enables an in-depth pharmacodynamic comparison between novel drug leads and may constitute an essential tool for the design of potential drug combinations.

Several quinoline derivatives incorporating arylnitro and aminochalcone moieties were synthesized and evaluated in vitro against a broad panel of trypanosomatid protozoan parasites responsible for sleeping sickness (Trypanosoma brucei rhodesiense), nagana (Trypanosoma brucei brucei), Chagas disease (Trypanosoma cruzi), and leishmaniasis (Leishmania infantum). Several of the compounds demonstrated significant antiprotozoal activity. Specifically, compounds 2c, 2d, and 4i displayed submicromolar activity against T. b. rhodesiense with half-maximal effective concentration (EC 50 ) values of 0.68, 0.8, and 0.19 µM, respectively, and with a high selectivity relative to human lung fibroblasts and mouse primary macrophages (∼100-fold). Compounds 2d and 4i also showed considerable activity against T. b. brucei with EC 50 values of 1.4 and 0.4 µM, respectively., (© 2024 The Authors. Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

The mosquito Aedes aegypti is distributed worldwide and is recognized as the primary vector for dengue in numerous countries. To investigate whether the fitness cost of a single DENV-1 isolate varies among populations, we selected four Ae. aegypti populations from distinct localities: Australia (AUS), Brazil (BRA), Pakistan (PAK), and Peru (PER). Utilizing simple methodologies, we concurrently assessed survival rates and fecundity. Overall, DENV-1 infection led to a significant decrease in mosquito survival rates, with the exception of the PER population. Furthermore, infected Ae. aegypti from PAK, the population with the lowest infection rate among those tested, exhibited a noteworthy reduction in egg laying. These findings collectively suggest that local mosquito-virus adaptations may influence dengue transmission in endemic settings.

Chagas disease (CD) is a parasitic neglected tropical disease (NTD) caused by the protozoan Trypanosoma cruzi that affects 6 million people worldwide, often resulting in financial burden, morbidity, and mortality in endemic regions. Given a lack of highly efficient and safe treatments, new, affordable, and fit-for-purpose drugs for CD are urgently needed. In this work, we present a hit-to-lead campaign for novel cyanopyridine analogues as antichagasic agents. In a phenotypic screening against intracellular T. cruzi , hits 1 and 2 were identified and displayed promising potency combined with balanced physicochemical properties. As part of the Lead Optimization Latin America consortium, a set of 40 compounds was designed, synthesized, and tested against T. cruzi intracellular amastigotes and relevant human cell lines. The structural modifications were focused on three positions: cyanopyridine core, linker, and right-hand side. The ADME properties of selected compounds, lipophilicity, kinetic solubility, permeability, and liver microsomal stability, were evaluated. Compounds 1-9 displayed good potency (EC 50 T. cruzi amastigote <1 μM), and most compounds did not present significant cytotoxicity (CC 50 MRC-5 = 32-64 μM). Despite the good balance between potency and selectivity, the antiparasitic activity of the series appeared to be driven by lipophilicity, making the progression of the series unfeasible due to poor ADME properties and potential promiscuity issues., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Herein, we report a series of 1,3-diarylpyrazoles that are analogues of compound 26 /HIT 8. We previously identified this molecule as a 'hit' during a high-throughput screening campaign for autophagy inducers. A variety of synthetic strategies were utilized to modify the 1,3-diarylpyrazole core at its 1-, 3-, and 4-position. Compounds were assessed in vitro to identify their cytotoxicity properties. Of note, several compounds in the series displayed relevant cytotoxicity, which warrants scrutiny while interpreting biological activities that have been reported for structurally related molecules. In addition, antiparasitic activities were recorded against a range of human-infective protozoa, including Trypanosoma cruzi , T. brucei rhodesiense , and Leishmania infantum . The most interesting compounds displayed low micromolar whole-cell potencies against individual or several parasitic species, while lacking cytotoxicity against human cells.

Addressing the challenges of quiescence and post-treatment relapse is of utmost importance in the microbiology field. This study shows that Leishmania infantum and L. donovani parasites rapidly enter into quiescence after an estimated 2-3 divisions in both human and mouse bone marrow stem cells. Interestingly, this behavior is not observed in macrophages, which are the primary host cells of the Leishmania parasite. Transcriptional comparison of the quiescent and non-quiescent metabolic states confirmed the overall decrease of gene expression as a hallmark of quiescence. Quiescent amastigotes display a reduced size and signs of a rapid evolutionary adaptation response with genetic alterations. Our study provides further evidence that this quiescent state significantly enhances resistance to treatment. Moreover, transitioning through quiescence is highly compatible with sand fly transmission and increases the potential of parasites to infect cells. Collectively, this work identified stem cells in the bone marrow as a niche where Leishmania quiescence occurs, with important implications for antiparasitic treatment and acquisition of virulence traits., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dirkx et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

N-Pyrazolylcarboxamides and N-pyrazolylureas represent promising lead compounds for the development of novel antileishmanial drugs. Herein, we report the late-stage diversification of 3-bromopyrazoles 10 A/B and 14 A by Pd-catalyzed Sonogashira and Suzuki-Miyaura cross coupling reactions. The electron-withdrawing properties of the cyano moiety in 4-position of the pyrazole ring limited the acylation of the primary amino moiety in 5-position. A large set of pyrazoles bearing diverse aryl and alkynyl substituents in 3-position was prepared and the antileishmanial and antitrypanosomal activity was recorded. The urea 38 lacking the electron withdrawing cyano moiety in 4-position and containing the large 4-benzylpiperidinoo moiety exhibited a modest antileishmanial (IC 50 =19 μM) and antitrypanosomal activity (IC 50 =7.9 μM)). However, its considerable toxicity against the PMM and MRC-5 cells indicates low selectivity, i. e. a small gap between the desired antiparasitic activity and undesired cytotoxicity of <2- to 4-fold., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Parasitic vector-borne diseases (VBDs) represent nearly 20% of the global burden of infectious diseases. Moreover, the spread of VBDs is enhanced by global travel, urbanization, and climate change. Treatment of VBDs faces challenges due to limitations of existing drugs, as the potential for side effects in nontarget species raises significant environmental concerns. Consequently, considering environmental risks early in drug development processes is critically important. Here, we examine the environmental risk assessment process for veterinary medicinal products in the European Union and identify major gaps in the ecotoxicity data of these drugs. By highlighting the scarcity of ecotoxicological data for commonly used antiparasitic drugs, we stress the urgent need for considering the One Health concept. We advocate for employing predictive tools and nonanimal methodologies such as New Approach Methodologies at early stages of antiparasitic drug research and development. Furthermore, adopting progressive approaches to mitigate ecological risks requires the integration of nonstandard tests that account for real-world complexities and use environmentally relevant exposure scenarios. Such a strategy is vital for a sustainable drug development process as it adheres to the principles of One Health, ultimately contributing to a healthier and more sustainable world.

Leishmania is a trypanosomatid parasite that causes skin lesions in its cutaneous form. Current therapies rely on old and expensive drugs, against which the parasites have acquired considerable resistance. Trypanosomatids are unable to synthesize purines relying on salvaging from the host, and nucleoside analogues have emerged as attractive antiparasitic drug candidates. 4-Methyl-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine (CL5564), an analogue of tubercidin in which the amine has been replaced by a methyl group, demonstrates activity against Trypanosoma cruzi and Leishmania infantum . Herein, we investigated its in vitro and in vivo activity against L. amazonensis . CL5564 was 6.5-fold ( P = 0.0002) more potent than milteforan™ (ML) against intracellular forms in peritoneal mouse macrophages, and highly selective, while combination with ML gave an additive effect. These results stimulated us to study the activity of CL5564 in mouse model of cutaneous Leishmania infection. BALB/c female and male mice infected by L. amazonensis treated with CL5564 (10 mg kg −1 , intralesional route for five days) presented a >93% reduction of paw lesion size likely ML given orally at 40 mg kg −1 , while the combination (10 + 40 mg kg −1 of CL5564 and ML, respectively) caused >96% reduction. The qPCR confirmed the suppression of parasite load, but only the combination approach reached 66% of parasitological cure. These results support additional studies with nucleoside derivatives.

Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment., (© 2024. The Author(s).)

Human African trypanosomiasis (HAT) still faces few therapeutic options and emerging drug resistance, stressing an urgency for novel antitrypanosomal drug discovery. Here, we describe lead optimization efforts aiming at improving antitrypanosomal efficacy and better physicochemical properties based on our previously reported optimized hit NPD-2975 (pIC 50 7.2). Systematic modification of the 5-phenylpyrazolopyrimidinone NPD-2975 led to the discovery of a R 4 -substituted analogue 31c (NPD-3519), showing higher in vitro potency (pIC 50 7.8) against Trypanosoma brucei and significantly better metabolic stability. Further, in vivo pharmacokinetic evaluation of 31c and experiments in an acute T. brucei mouse model confirmed improved oral bioavailability and antitrypanosomal efficacy at 50 mg/kg with no apparent toxicity. With good physicochemical properties, low toxicity, improved pharmacokinetic features, and in vivo efficacy, 31c may serve as a promising candidate for future drug development for HAT.

A library of imidazo[1,2-a]pyridine-appended chalcones were synthesized and characterized using 1 H NMR, 13 C NMR and HRMS. The synthesized analogues were screened for their antikinetoplastid activity against Trypanosoma cruzi, Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense and Leishmania infantum. The analogues were also tested for their cytotoxicity activity against human lung fibroblasts and primary mouse macrophages. Among all screened derivatives, 7f was found to be the most active against T. cruzi and T. b. brucei exhibiting IC 50 values of 8.5 and 1.35 μM, respectively. Against T. b. rhodesiense, 7e was found to be the most active with an IC 50 value of 1.13 μM. All synthesized active analogues were found to be non-cytotoxic against MRC-5 and PMM with selectivity indices of up to more than 50., (© 2023 The Authors. Chemical Biology & Drug Design published by John Wiley & Sons Ltd.)

Various purine-based nucleoside analogues have demonstrated unexpected affinity for nonpurinergic G protein-coupled receptors (GPCRs), such as opioid and serotonin receptors. In this work, we synthesized a small library of new 7-deazaadenosine and pyrazolo[3,4- d ]pyrimidine riboside analogues, featuring dual C7 and N 6 modifications and assessed their affinity for various GPCRs. During the course of the synthesis of 7-ethynyl pyrazolo[3,4- d ]pyrimidine ribosides, we observed the formation of an unprecedented tricyclic nucleobase, formed via a 6- endo-dig ring closure. The synthesis of this tricyclic nucleoside was optimized, and the substrate scope for such cyclization was further explored because it might avail further exploration in the nucleoside field. From displacement experiments on a panel of GPCRs and transporters, combining C7 and N 6 modifications afforded noncytotoxic nucleosides with micromolar and submicromolar affinity for different GPCRs, such as the 5-hydroxytryptamine (5-HT) 2B , κ-opioid (KOR), and σ 1/2 receptor. These results corroborate that the novel nucleoside analogues reported here are potentially useful starting points for the further development of modulators of GPCRs and transmembrane proteins., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

New drugs for visceral leishmaniasis that are safe, low cost, and adapted to the field are urgently required. Despite concerted efforts over the last several years, the number of new chemical entities that are suitable for clinical development for the treatment of Leishmania remains low. Here, we describe the discovery and preclinical development of DNDI-6174, an inhibitor of Leishmania cytochrome bc 1 complex activity that originated from a phenotypically identified pyrrolopyrimidine series. This compound fulfills all target candidate profile criteria required for progression into preclinical development. In addition to good metabolic stability and pharmacokinetic properties, DNDI-6174 demonstrates potent in vitro activity against a variety of Leishmania species and can reduce parasite burden in animal models of infection, with the potential to approach sterile cure. No major flags were identified in preliminary safety studies, including an exploratory 14-day toxicology study in the rat. DNDI-6174 is a cytochrome bc 1 complex inhibitor with acceptable development properties to enter preclinical development for visceral leishmaniasis.

Background: Visceral leishmaniasis (VL), a life-threatening neglected tropical disease, is targeted for elimination from Nepal by the year 2026. The national VL elimination program is still confronted with many challenges including the increasingly widespread distribution of the disease over the country, local resurgence and the questionable efficacy of the key vector control activities. In this study, we assessed the status and risk of Leishmania donovani transmission based on entomological indicators including seasonality, natural Leishmania infection rate and feeding behavior of vector sand flies, Phlebotomus argentipes, in three districts that had received disease control interventions in the past several years in the context of the disease elimination effort., Methods: We selected two epidemiologically contrasting settings in each survey district, one village with and one without reported VL cases in recent years. Adult sand flies were collected using CDC light traps and mouth aspirators in each village for 12 consecutive months from July 2017 to June 2018. Leishmania infection was assessed in gravid sand flies targeting the small-subunit ribosomal RNA gene of the parasite (SSU-rRNA) and further sequenced for species identification. A segment (~ 350 bp) of the vertebrate cytochrome b (cytb) gene was amplified from blood-fed P. argentipes from dwellings shared by both humans and cattle and sequenced to identify the preferred host., Results: Vector abundance varied among districts and village types and peaks were observed in June, July and September to November. The estimated Leishmania infection rate in vector sand flies was 2.2% (1.1%-3.7% at 95% credible interval) and 0.6% (0.2%-1.3% at 95% credible interval) in VL and non-VL villages respectively. The common source of blood meal was humans in both VL (52.7%) and non-VL (74.2%) villages followed by cattle., Conclusions: Our findings highlight the risk of ongoing L. donovani transmission not only in villages with VL cases but also in villages not reporting the presence of the disease over the past several years within the districts having disease elimination efforts, emphasize the remaining threats of VL re-emergence and inform the national program for critical evaluation of disease elimination strategies in Nepal., (© 2023. The Author(s).)

Trypanosomes and Leishmania are parasitic protozoans that affect millions of people globally. Herein we report the synthesis of 2-aroyl quinazolinones and their antiprotozoal efficacy against Trypanosoma brucei, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania infantum. These compounds were counter-screened against a human cell line for cytotoxicity. Thirteen of the twenty target compounds in this study inhibited the growth of these parasites, with compounds KJ1, and KJ10 exhibiting IC 50 values of 4.7 μM (T. b. brucei) and 1.1 μM (T. b. rhodesiense), respectively., (© 2023 The Authors. Chemical Biology & Drug Design published by John Wiley & Sons Ltd.)

Dipeptidyl peptidase 9 (DPP9) is a proline-selective serine protease that plays a key role in NLRP1- and CARD8-mediated inflammatory cell death (pyroptosis). No selective inhibitors have hitherto been reported for the enzyme: all published molecules have grossly comparable affinities for DPP8 and 9 because of the highly similar architecture of these enzymes' active sites. Selective DPP9 inhibitors would be highly instrumental to address unanswered research questions on the enzyme's role in pyroptosis, and they could also be investigated as therapeutics for acute myeloid leukemias. Compounds presented in this manuscript ( 42 and 47 ) combine low nanomolar DPP9 affinities with unprecedented DPP9-to-DPP8 selectivity indices up to 175 and selectivity indices >1000 toward all other proline-selective proteases. To rationalize experimentally obtained data, a molecular dynamics study was performed. We also provide in vivo pharmacokinetics data for compound 42 .

Leishmaniasis is a collection of diseases caused by more than 20 Leishmania parasite species that manifest as either visceral, cutaneous, or mucocutaneous leishmaniasis. Despite the significant mortality and morbidity associated with leishmaniasis, it remains a neglected tropical disease. Existing treatments have variable efficacy, significant toxicity, rising resistance, and limited oral bioavailability, which necessitates the development of novel and affordable therapeutics. Here, we report on the continued optimization of a series of imidazopyridines for visceral leishmaniasis and a scaffold hop to a series of substituted 2-(pyridin-2-yl)-6,7-dihydro-5 H -pyrrolo[1,2- a ]imidazoles with improved absorption, distribution, metabolism, and elimination properties.

Human African Trypanosomiasis (HAT), caused by Trypanosoma brucei , is one of the neglected tropical diseases with a continuing need for new medication. We here describe the discovery of 5-phenylpyrazolopyrimidinone analogs as a novel series of phenotypic antitrypanosomal agents. The most potent compound, 30 (NPD-2975), has an in vitro IC 50 of 70 nM against T. b. brucei with no apparent toxicity against human MRC-5 lung fibroblasts. Showing good physicochemical properties, low toxicity potential, acceptable metabolic stability, and other pharmacokinetic features, 30 was further evaluated in an acute mouse model of T. b. brucei infection. After oral dosing at 50 mg/kg twice per day for five consecutive days, all infected mice were cured. Given its good drug-like properties and high in vivo antitrypanosomal potential, the 5-phenylpyrazolopyrimidinone analog 30 represents a promising lead for future drug development to treat HAT.

Malaria continues to pose a significant health threat, causing thousands of deaths each year. The limited availability of vaccines and medications, combined with the emergence of drug resistance, further complicates the fight against this disease. In this study, we aimed to enhance the antimalarial potency of the previously reported hit compound BIPPO (pIC 50 5.9). Through systematic modification of pyrazolopyrimidinone analogs, we discovered the promising analog 30 (NPD-3547), which exhibited approximately one log unit higher in vitro potency (pIC 50 6.8) against Plasmodium falciparum . Furthermore, we identified several other BIPPO analogs ( 23 , 28 , 29 and 47a ) with potent antimalarial activity (pIC 50 > 6.0) and favorable metabolic stability in mouse liver microsomes. These compounds can serve as new tools for further optimization towards the development of potential candidates for antimalarial studies.

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.

African Animal Trypanosomiasis (AAT), caused predominantly by Trypanosoma brucei brucei , T. vivax and T. congolense , is a fatal livestock disease throughout Sub-Saharan Africa. Treatment options are very limited and threatened by resistance. Tubercidin (7-deazaadenosine) analogs have shown activity against individual parasites but viable chemotherapy must be active against all three species. Divergence in sensitivity to nucleoside antimetabolites could be caused by differences in nucleoside transporters. Having previously characterized the T. brucei nucleoside carriers, we here report the functional expression and characterization of the main adenosine transporters of T. vivax (TvxNT3) and T. congolense (TcoAT1/NT10), in a Leishmania mexicana cell line ('SUPKO') lacking adenosine uptake. Both carriers were similar to the T. brucei P1-type transporters and bind adenosine mostly through interactions with N3, N7 and 3'-OH. Expression of TvxNT3 and TcoAT1 sensitized SUPKO cells to various 7-substituted tubercidins and other nucleoside analogs although tubercidin itself is a poor substrate for P1-type transporters. Individual nucleoside EC 50 s were similar for T. b. brucei , T. congolense , T. evansi and T. equiperdum but correlated less well with T. vivax . However, multiple nucleosides including 7-halogentubercidines displayed pEC50>7 for all species and, based on transporter and anti-parasite SAR analyses, we conclude that nucleoside chemotherapy for AAT is viable.

Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi. Because current treatments present several limitations, including long duration, variable efficacy and serious side effects, there is an urgent need to explore new antitrypanosomal drugs. The present study describes the hit-to-lead optimization of a 2-aminobenzimidazole hit 1 identified through in vitro phenotypic screening of a chemical library against intracellular Trypanosoma cruzi amastigotes, which focused on optimizing potency, selectivity, microsomal stability and lipophilicity. Multiparametric Structure-Activity Relationships were investigated using a set of 277 derivatives. Although the physicochemical and biological properties of the initial hits were improved, a combination of low kinetic solubility and in vitro cytotoxicity against mammalian cells prevented progression of the best compounds to an efficacy study using a mouse model of Chagas disease., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Luiz Carlos Dias reports financial support was provided by Fundação de Amparo a Pesquisa do Estado de São Paulo - Fapesp., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Fosmidomycin is a natural antibiotic with potent 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 most bacteria, including A. baumanii and M. tuberculosis, and apicomplexan parasites, including Plasmodium parasites. Mainly as a result of its high polarity, fosmidomycin displays suboptimal pharmacokinetic properties. Furthermore, fosmidomycin is inactive against A. baumannii and 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 on the expansion of the acyloxymethyl and alkoxycarbonyloxymethyl phosphonate ester prodrug series of a fosmidomycin surrogate. Prodrug promoieties were designed based on electronic, lipophilic and siderophoric properties. This investigation led to the discovery of derivatives with two-digit nanomolar and submicromolar IC 50 -values against P. falciparum and A. baumanii, respectively., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

The application of in vivo bioluminescent imaging in infectious disease research has significantly increased over the past years. The detection of transgenic parasites expressing wildtype firefly luciferase is however hampered by a relatively low and heterogeneous tissue penetrating capacity of emitted light. Solutions are sought by using codon-optimized red-shifted luciferases that yield higher expression levels and produce relatively more red or near-infrared light, or by using modified bioluminescent substrates with enhanced cell permeability and improved luminogenic or pharmacokinetic properties. In this study, the in vitro and in vivo efficacy of two modified bioluminescent substrates, CycLuc1 and AkaLumine-HCl, were compared with that of D-luciferin as a gold standard. Comparisons were made in experimental and insect-transmitted animal models of leishmaniasis (caused by intracellular Leishmania species) and African trypanosomiasis (caused by extracellular Trypanosoma species), using parasite strains expressing the red-shifted firefly luciferase PpyRE9. Although the luminogenic properties of AkaLumine-HCl and D-luciferin for in vitro parasite detection were comparable at equal substrate concentrations, AkaLumine-HCl proved to be unsuitable for in vivo infection follow-up due to high background signals in the liver. CycLuc1 presented a higher in vitro luminescence compared to the other substrates and proved to be highly efficacious in vivo, even at a 20-fold lower dose than D-luciferin. This efficacy was consistent across infections with the herein included intracellular and extracellular parasitic organisms. It can be concluded that CycLuc1 is an excellent and broadly applicable alternative for D-luciferin, requiring significantly lower doses for in vivo bioluminescent imaging in rodent models of leishmaniasis and African trypanosomiasis.

Advanced nanoscale antimicrobials, originated from the combination of noble metal nanoparticles (NPs) with conventional antimicrobial 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 β-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 complementary spectroscopic techniques, such as UV-vis, ζ-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., (Copyright © 2022, Hada et al.)

Approximately 20% of sleeping sickness patients exhibit respiratory complications, however, with a largely unknown role of the parasite. Here we show that tsetse fly-transmitted Trypanosoma brucei parasites rapidly and permanently colonize the lungs and occupy the extravascular spaces surrounding the blood vessels of the alveoli and bronchi. They are present as nests of multiplying parasites exhibiting close interactions with collagen and active secretion of extracellular vesicles. The local immune response shows a substantial increase of monocytes, macrophages, dendritic cells and γδ and activated αβ T cells and a later influx of neutrophils. Interestingly, parasite presence results in a significant reduction of B cells, eosinophils and natural killer cells. T. brucei infected mice show no infection-associated pulmonary dysfunction, mirroring the limited pulmonary clinical complications during sleeping sickness. However, the substantial reduction of the various immune cells may render individuals more susceptible to opportunistic infections, as evident by a co-infection experiment with respiratory syncytial virus. Collectively, these observations provide insights into a largely overlooked target organ, and may trigger new diagnostic and supportive therapeutic approaches for sleeping sickness., (© 2022. The Author(s).)

Microglia and border-associated macrophages (BAMs) are brain-resident self-renewing cells. Here, we examined the fate of microglia, BAMs, and recruited macrophages upon neuroinflammation and through resolution. Upon infection, Trypanosoma brucei parasites invaded the brain via its border regions, triggering brain barrier disruption and monocyte infiltration. Fate mapping combined with single-cell sequencing revealed microglia accumulation around the ventricles and expansion of epiplexus cells. Depletion experiments using genetic targeting revealed that resident macrophages promoted initial parasite defense and subsequently facilitated monocyte infiltration across brain barriers. These recruited monocyte-derived macrophages outnumbered resident macrophages and exhibited more transcriptional plasticity, adopting antimicrobial gene expression profiles. Recruited macrophages were rapidly removed upon disease resolution, leaving no engrafted monocyte-derived cells in the parenchyma, while resident macrophages progressively reverted toward a homeostatic state. Long-term transcriptional alterations were limited for microglia but more pronounced in BAMs. Thus, brain-resident and recruited macrophages exhibit diverging responses and dynamics during infection and resolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)