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Sleeping sickness and leishmaniasis are neglected tropical diseases that threaten millions of people. The currently available therapies present several limitations, including high toxicity, lack of efficacy, and emerging drug resistance, prompting a search for novel therapeutic agents. In this work, we designed, synthesized, and in vitro evaluated the activity of new pyrimido[5,4-d]pyrimidines against Trypanosoma brucei and Leishmania infantum (promastigote and amastigote forms). The cytotoxicity of the compounds against the THP1 cell line was also assessed. Most tested compounds presented low micromolar activity against T. brucei with IC50 values in the range between 0.9 and 13.4 mu M, and one compound also showed activity against L. infantum (IC50 = 3.13 mu M). Several molecules presented a selectivity index higher than 10. The most active compound against booth parasites is derivative 4c, with IC50 = 0.94 mu M (SI > 107) against T. brucei and IC50 = 3.13 mu M (SI > 32) against L. infantum. This data enabled the identification of a new promising molecular scaffold for developing a novel class of antitrypanosomal and antileishmanial agents., This work and N.S. were supported by Fundacao para a Ciencia e Tecnologia (FCT)/Ministerio da Educacao e Cieencia (MEC) cofunded by the FEDER through the COMPETE 2020 -Operacional Programme for Competitiveness and Internationalisation (POCI) ref POCI-01-0145-FEDER-031013 and funds in the framework of the Strategic Funding of CQUM (UID/QUI/00686/2020), (UID/QUI/00686/2018) and Rede Nacional de RMN (PINFRA/22161/2016).

The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure-activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei, whilst retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

Dogs are highly valued companions and work animals that are susceptible to many life-threatening conditions such as canine leishmaniosis (CanL). Plasma-derived extracellular vesicles (EVs), exploited extensively in biomarker discovery, constitute a mostly untapped resource in veterinary sciences. Thus, the definition of proteins associated with plasma EVs recovered from healthy and diseased dogs with a relevant pathogen would be important for biomarker development. For this, we recovered, using size-exclusion chromatography (SEC), EVs from 19 healthy and 20 CanL dogs’ plasma and performed proteomic analysis by LC-MS/MS to define their core proteomic composition and search for CanL-associated alterations. EVs-specific markers were identified in all preparations and also non-EVs proteins. Some EVs markers such as CD82 were specific to the healthy animals, while others, such as the Integrin beta 3 were identified in most samples. The EVs-enriched preparations allowed the identification of 529 canine proteins that were identified in both groups, while 465 and 154 were only identified in healthy or CanL samples, respectively. A GO enrichment analysis revealed few CanL-specific terms. Leishmania spp. protein identifications were also found, although with only one unique peptide. Ultimately, CanL-associated proteins of interest were identified and a core proteome was revealed that will be available for intra- and inter-species comparisons.

Funding text This research was funded by the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was also supported by the Associate Laboratory for Green Chemistry LAQV financed by national funds from FCT/MCTES (UIDB/50006/2020, UIDP/50006/2020 and LA/P/0008/2020). The authors also thank Fundação para a Ciência e Tecnologia for the projects PTDC/EAM-AMB/2023/2021, PTDC/QUI-QOR/32406/2017 and PTDC/BTM-SAL/29786/2017. N.S. was supported by PTDC/SAU-PAR/31013/2017. A.F.M.S and Ž.P. also acknowledge FCT-MCTES for the PhD Grant (SFRH/BD/132551/2017) and for the Norma Transitória DL 57/2016 Program contract, respectively. The NMR spectrometers are part of The National NMR Facility, supported by FCT (ROTEIRO/0031/2013 - PINFRA/22161/2016) (co-financed by FEDER through COMPETE 2020, POCI, and PORL and FCT through PIDDAC). Nowadays, organic salts and ionic liquids (OSILs) containing active pharmaceutical ingredients (APIs) are being explored as drug delivery systems in modern therapies (OSILs-API). In that sense, this work is focused on the development of novel OSILs-API based on amphotericin B through an innovative procedure and the evaluation of the respective biological activity against Leishmania infantum. Several ammonium, methylimidazolium, pyridinium and phosphonium organic cations combined with amphotericin B as anion were synthesized in moderate to high yields and high purities by the water-reduced buffer neutralization method. All prepared compounds were characterized to confirm the desired chemical structure and the specific optical rotation ([α]D25) was also determined. The biological assays performed on L. infantum promastigotes showed increased activity against this parasitic disease when compared with the starting chloride forms and amphotericin B alone, highlighting [P6,6,6,14][AmB] as the most promising formulation. Possible synergism in the antiprotozoal activity was also evaluated for [P6,6,6,14][AmB], since it was proven to be the compound with the highest toxicity. This work reported a simple synthetic method, which can be applied to prepare other organic salts based on molecules containing fragile chemical groups, demonstrating the potential of these OSILs-AmB as possible agents against leishmaniasis. publishersversion published

Nowadays, organic salts and ionic liquids (OSILs) containing active pharmaceutical ingredients (APIs) are being explored as drug delivery systems in modern therapies (OSILs-API). In that sense, this work is focused on the development of novel OSILs-API based on amphotericin B through an innovative procedure and the evaluation of the respective biological activity against

Canine leishmaniosis (CanL) is a vector-borne disease caused by

Leishmaniasis consists of a group of neglected diseases, which affect millions of people worldwide. First- and second-line chemotherapies, which present high toxicity and variable efficacy, have been available for decades leading to the appearance of resistance. Globally, volatile oils have received much attention due to their increase use as effective bioactive compounds in folk medicine. They have been employed against Leishmania parasites with a positive impact in the treatment of this neglected disease. In this review, the leishmanicidal activity of volatile oils and their isolates have been described as promising novel candidate molecules for the development of leishmanicidal drugs. The purpose of this review is to critically discuss several works published between 2000 and 2020 that have described the utilization of volatile oils and some of their active principles from diverse plant sources against different species of Leishmania.

Malaria is a major public health concern, as a highly effective human vaccine remains elusive. The efficacy of a subunit vaccine targeting the most abundant protein of the sporozoite surface, the circumsporozoite protein (CSP) has been hindered by difficulties in generating an effective humoral response in both quantity and quality. Using the rodent Plasmodium yoelii model we report here that immunization with CSP adjuvanted with 5’ppp-dsRNA, a RIG-I agonist, confers early and long-lasting sterile protection in mice against stringent sporozoite and mosquito bite challenges. The immunization induced high levels of antibodies, which were functional in targeting and killing the sporozoites and were sustained over time through the accumulation of long-lived plasma cells in the bone marrow. Moreover, 5’ppp-dsRNA-adjuvanted immunization with the CSP of P. falciparum was also significantly protective against challenges using a transgenic PfCSP-expressing P. yoelii parasite. Conversely, using the TLR3 agonist poly(A:U) as adjuvant resulted in a formulation that despite inducing high antibody levels was unable to generate equally functional antibodies and was, consequently, less protective. In conclusion, we demonstrate that using 5’ppp-dsRNA as an adjuvant to vaccines targeting CSP induces effective anti-Plasmodium humoral immunity.

Sleeping sickness and leishmaniasis are neglected tropical diseases that threaten millions of people. The currently available therapies present several limitations, including high toxicity, lack of efficacy, and emerging drug resistance, prompting a search for novel therapeutic agents. In this work, we designed, synthesized, and

Leishmaniases are neglected tropical diseases. The treatment of leishmaniasis relies exclusively on chemotherapy including amphotericin B (AmB), miltefosine (hexadecylphosphocholine), and pentamidine. Besides the fact that these molecules are harmful for patients, little is known about the impact of such antileishmanial drugs on primary human cells in relation to immune function. The present study demonstrates that all antileishmanial drugs inhibit CD4 and CD8 T cell proliferation at the doses that are not related to increased cell death. Our results highlight that antileishmanial drugs have an impact on monocytes by altering the expression of IL-12 induced by LPS, whereas only AmB induced IL-10 secretion; both cytokines are essential in regulating Th1 cell–mediated immunity. Interestingly, IL-12 and anti–IL-10 Abs improved T cell proliferation inhibited by AmB. Furthermore, our results show that in contrast to hexadecylphosphocholine and pentamidine, AmB induced gene expression of the inflammasome pathway. Thus, AmB induced IL-1β and IL-18 secretions, which are reduced by specific inhibitors of caspase activation (Q-VD) and NLRP3 activation (MCC950). Our results reveal previously underestimated effects of antileishmanial drugs on primary human cells.

Trypanosomiasis is a parasitic disease, affecting both humans and animals, in the form of Human African Trypanosomiasis and Nagana disease, respectively. Anemia is one of the most common symptoms of trypanosomiasis, and if left unchecked, can cause severe complications and even death. Several factors have been associated with the development of this anemia, including dysregulation of iron homeostasis, but little is known about the molecular mechanisms involved. Here, using murine models, we study the involvement of hepcidin, the key regulator of iron metabolism and an important player in the development of anemia of inflammation. Our data shows two stages for the progression of anemia, to which hepcidin contributes: a first stage, when anemia develops, with a likely cytokine-mediated stimulation of hepcidin and subsequent limitation in iron availability and erythropoiesis, and a second stage, of recovery, where hepcidin elevation declines due to the decreased inflammatory signal and increased production of erythroid regulators by the kidney, spleen and bone marrow, thus leading to an increase in iron release and availability and enhanced erythropoiesis. In agreement, in hepcidin knockout mice, anaemia is much milder and its recovery is complete, in contrast to wild-type animals which do not fully recover from anaemia after 21 days. Aside from all other factors known to be involved in the development of anemia during trypanosomiasis, there clearly is an important contribution of hepcidin for both its development and recovery.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00589. Malaria, leishmaniasis, and sleeping sickness are potentially fatal diseases that represent a real health risk for more than 3,5 billion people. New antiparasitic compounds are urgent leading to a constant search for novel scaffolds. Herein, pyrazino[2,1-b]quinazoline-3,6-diones containing indole alkaloids were explored for their antiparasitic potential against Plasmodium falciparum, Trypanosoma brucei, and Leishmania infantum. The synthetic libraries furnished promising hit compounds that are species specific (7, 12) or with broad antiparasitic activity (8). Structure-activity relationships were more evident for Plasmodium with anti-isomers (1S,4R) possessing excellent antimalarial activity, while the presence of a substituent on the anthranilic acid moiety had a negative effect on the activity. Hit compounds against malaria did not inhibit β-hematin, and in silico studies predicted these molecules as possible inhibitors for prolyl-tRNA synthetase both from Plasmodium and Leishmania. These results disclosed a potential new chemotype for further optimization toward novel and affordable antiparasitic drugs. publishersversion published

Human African Trypanosomiasis (HAT, sleeping sickness) and Animal African Trypanosomiasis (AAT) are neglected tropical diseases generally caused by the same etiological agent, Trypanosoma brucei. Despite important advances in the reduction or disappearance of HAT cases, AAT represents a risky reservoir of the infections. There is a strong need to control AAT, as is claimed by the European Commission in a recent document on the reservation of antimicrobials for human use. Control of AAT is considered part of the One Health approach established by the FAO program against African Trypanosomiasis. Under the umbrella of the One Health concepts, in this work, by analyzing the pharmacological properties of the therapeutic options against Trypanosoma brucei spp., we underline the need for clearer and more defined guidelines in the employment of drugs designed for HAT and AAT. Essential requirements are addressed to meet the challenge of drug use and drug resistance development. This approach shall avoid inter-species cross-resistance phenomena and retain drugs therapeutic activity.

Leishmaniasis is an important vector-borne neglected tropical disease caused by Leishmania parasites. Current anti-Leishmania chemotherapy is unsatisfactory, justifying the continued search for alternative treatment options. Herein, we demonstrate that luciferase-expressing Leishmania infantum axenic amastigotes, unlike promastigotes, are highly infectious to BALB/c mice and thus generate a robust bioluminescent signal in target organs, such as the liver and the spleen, as early as two weeks after infection. Treatment with the reference drugs amphotericin B and miltefosine was effective at reducing parasite burdens. This model allows the assessment of treatment efficacy using whole-mouse bioluminescence imaging without the need to wait several weeks for spleen infections to be detectable by this non-invasive method. In conclusion, we propose the use of this model in an initial approach to evaluate the treatment efficacy of promising chemical entities without having to sacrifice large numbers of animals or to wait several days for a readout. We thank Carla Oliveira from i3S for the support with the statistical analysis. Part of the content of this manuscript has been released as a pre-print in BioRxiv (https://doi.org/10.1101/326355). This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-031013 (PTDC/SAU-PAR/31013/2017). This work also received funds from: Norte-01-0145-FEDER-000012 “Structured program on bioengineered therapies for infectious diseases and tissue regeneration” of Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and through the Research Unit No. 4293; Individual funding from FCT through SFRH/BD/123734/2016 (to DC), SFRH/BD/121252/2016 (to PC) and CEECIND/02362/2017 (to JT).

Blood-sucking arthropods transmit a variety of human pathogens acting as disseminators of the so-called vector-borne diseases. Leishmaniasis is a spectrum of diseases caused by different Leishmania species, transmitted quasi worldwide by sand flies. However, whereas many laboratories focus on the disease(s) and etiological agents, considerably less study the respective vectors. In fact, information on sand flies is neither abundant nor easy to find; aspects including basic biology, ecology, and sand-fly-Leishmania interactions are usually reported separately. Here, we compile elemental information on sand flies, in the context of leishmaniasis. We discuss the biology, distribution, and life cycle, the blood-feeding process, and the Leishmania-sand fly interactions that govern parasite transmission. Additionally, we highlight some outstanding questions that need to be answered for the complete understanding of parasite–vector–host interactions in leishmaniasis.

Leishmaniasis, a vector-borne parasitic protozoan disease, is among the most important neglected tropical diseases. In the absence of vaccines, disease management is challenging. The available chemotherapy is suboptimal, and there are growing concerns about the emergence of drug resistance. Thus, a better understanding of parasite biology is essential to generate new strategies for disease control. In this context, in vitro parasite exoproteome characterization enabled the identification of proteins involved in parasite survival, pathogenesis, and other biologically relevant processes. After 2005, with the availability of genomic information, these studies became increasingly feasible and revealed the true complexity of the parasite exoproteome. After the discovery of Leishmania extracellular vesicles (EVs), most exoproteome studies shifted to the characterization of EVs. The non-EV portion of the exoproteome, named the vesicle-depleted exoproteome (VDE), has been mostly ignored even if it accounts for a significant portion of the total exoproteome proteins. Herein, we summarize the importance of total exoproteome studies followed by a special emphasis on the available information and the biological relevance of the VDE. Finally, we report on how VDE can be studied and disclose how it might contribute to providing biologically relevant targets for diagnosis, drug, and vaccine development.

Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis, fatal if untreated. Vaccination is the most cost-effective approach to disease control; however, to date, no vaccines against human VL have been made available. This work examines the efficacy of a novel vaccine consisting of the Leishmania membrane protein KMP11, LEISH-F3+ (a recombinant fusion protein, composed of epitopes of the parasite proteins nucleoside hydrolase, sterol-24-c-methyltransferase, and cysteine protease B), and the sand fly salivary protein LJL143, in two dose ratios. The inclusion of the TLR4 agonist GLA-SE as an adjuvant, and the use of virosomes (VS) as a delivery system, are also examined. In a hamster model of VL, the vaccine elicited antigen-specific immune responses prior to infection with Leishmania infantum. Of note, the responses were greater when higher doses of KMP11 and LEISH-F3+ proteins were administered along with the GLA-SE adjuvant and/or when delivered within VS. Remarkably, hamsters immunized with the complete combination (i.e., all antigens in VS + GLA-SE) showed significantly lower parasite burdens in the spleen compared to those in control animals. This protection was underpinned by a more intense, specific humoral response against the KMP11, LEISH-F3+, and LJL143 antigens in vaccinated animals, but a significantly less intense antibody response to the pool of soluble Leishmania antigens (SLA). Overall, these results indicate that this innovative vaccine formulation confers protection against L. infantum infection, supporting the advancement of the vaccine formulation into process development and manufacturing and the conduction of toxicity studies towards future phase I human clinical trials, European Community’s Seventh Framework Programme, grant number 603181 (Clinical Studies on a Multivalent Vaccine for Human Visceral Leishmaniasis [MuLeVaClin]), and by the RD16CIII/0003/0002 and RD16/0027/0008 Red de Investigación Cooperativa de Enfermedades Tropicales, Subprograma RETICS del Plan Estatal de I+D+I 2013–2016, co-funded by ERDF

The optimization of compounds with multiple targets in the drug discovery cycle is a difficult multidimensional problem. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Efficient microwave-assisted synthesis of pteridines along with iterations of crystallographic structure determination were used to validate computational docking predictions and support derivation of a structure-activity relationship for multitarget inhibition. This approach yielded compounds showing picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, along with selective submicromolar inhibition of parasitic dihydrofolate reductase (DHFR). Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei, whilst retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

[Image: see text] Malaria, leishmaniasis, and sleeping sickness are potentially fatal diseases that represent a real health risk for more than 3,5 billion people. New antiparasitic compounds are urgent leading to a constant search for novel scaffolds. Herein, pyrazino[2,1-b]quinazoline-3,6-diones containing indole alkaloids were explored for their antiparasitic potential against Plasmodium falciparum, Trypanosoma brucei, and Leishmania infantum. The synthetic libraries furnished promising hit compounds that are species specific (7, 12) or with broad antiparasitic activity (8). Structure–activity relationships were more evident for Plasmodium with anti-isomers (1S,4R) possessing excellent antimalarial activity, while the presence of a substituent on the anthranilic acid moiety had a negative effect on the activity. Hit compounds against malaria did not inhibit β-hematin, and in silico studies predicted these molecules as possible inhibitors for prolyl-tRNA synthetase both from Plasmodium and Leishmania. These results disclosed a potential new chemotype for further optimization toward novel and affordable antiparasitic drugs.

A library of seventeen novel ether phospholipid analogues, containing 5-membered heterocyclic rings (1,2,3-triazolyl, isoxazolyl, 1,3,4-oxadiazolyl and 1,2,4-oxadiazolyl) in the lipid portion were designed and synthesized aiming to identify optimised miltefosine analogues. The compounds were evaluated for their in vitro antiparasitic activity against Leishmania infantum and Leishmania donovani intracellular amastigotes, against Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the substituents of the heterocyclic ring (tail) and the oligomethylene spacer between the head group and the heterocyclic ring was found to affect the activity and toxicity of these compounds leading to a significantly improved understanding of their structure–activity relationships. The early ADMET profile of the new derivatives did not reveal major liabilities for the potent compounds. The 1,2,3-triazole derivative 27 substituted by a decyl tail, an undecyl spacer and a choline head group exhibited broad spectrum antiparasitic activity. It possessed low micromolar activity against the intracellular amastigotes of two L. infantum strains and T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes, while its cytotoxicity concentration (CC50) against THP-1 macrophages ranged between 50 and 100 μM. Altogether, our work paves the way for the development of improved ether phospholipid derivatives to control neglected tropical diseases.

Funding from the European Community's Seventh Framework Programme under grant agreements No. 602773 (Project KINDRED) was received for all partners in this work. This work also received funds from FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior through the Research Unit No. 4293 and project POCI-01-0145-FEDER-031013 (PTDC/SAUPAR/31013/2017 to NS); Individual funding from FCT through SFRH/BD/133485/2017 (to MS) and CEECIND/02362/2017 (to JT). Neglected tropical diseases caused by kinetoplastid parasites (Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp.) place a significant health and economic burden on developing nations worldwide. Current therapies are largely out-dated, inadequate and facing mounting drug resistance from the causative parasites. Thus, there is an urgent need for drug discovery and development. Target-led drug discovery approaches have focused on the identification of parasite enzymes catalysing essential biochemical processes, which significantly differ from equivalent proteins found in humans, thereby providing potentially exploitable therapeutic windows. One such target is ribose 5-phosphate isomerase B (RpiB), an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, which catalyses the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Although protozoan RpiB has been the focus of numerous targeted studies, compounds capable of selectively inhibiting this parasite enzyme have not been identified. Here, we present the results of a fragment library screening against Leishmania infantum RpiB, performed using thermal shift analysis. Hit fragments were shown to be effective inhibitors of LiRpiB in activity assays, and several were capable of selectively inhibiting parasite growth in vitro. These results support the identification of LiRpiB as a validated therapeutic target. The X-ray crystal structure of apo LiRpiB was also solved, permitting docking studies to assess how hit fragments might interact with LiRpiB to inhibit its activity. Overall, this work will guide structure-based development of LiRpiB inhibitors as anti-leishmanial agents. Postprint

Drugs that target human thymidylate synthase (hTS) are widely used in anti-cancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces its over-expression and the development of drug resistance. We thus pursued an alternative strategy that led to the discovery of TS-dimer disrupters that bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. We performed a structural, spectroscopic and kinetic investigation of the effects of these small molecules andthe best one, E7, accelerates the proteasomal degradation of hTS in cancer cells. E7 showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.

A proper description of the immune response to SARS-CoV-2 will be critical for the assessment of protection elicited after both infection and vaccination. Uncoupled T and B cell responses have been described in acute and convalescent patients and exposed individuals. We assessed the potential usefulness of whole blood stimulation assays to identify functional cellular immune responses to SARS-CoV-2. Blood from COVID-19 recovered individuals (5 months after infection) and negative subjects was stimulated for 24 hours with HLA predicted peptide “megapools” of the Spike and Nucleoprotein, or the mixture of them. After stimulation, cytokines were quantified using a beads-based multiplex assay. Interleukin-2 and IFN-γ were found to be specific biomarkers of SARS-CoV-2 cellular response. Using the Spike and Nucleoprotein mixture, 91.3% of COVID-19 recovered individuals presented an IL-2 stimulation index over the cut-off, while 82.6% showed IFN-γ. All the negative individuals presented an IL-2 response under the cut-off, while 5.3% of these subjects presented positive IFN-γ stimulation indexes. Moreover, IL-2 production correlated with IgG levels for Spike 1, RBD, and Nucleocapsid. In conclusion, we demonstrate the potential of whole blood stimulation assays and the quantification of IL-2 and IFN-γ for the analysis of SARS-CoV-2 functional cellular responses.

Intracellular protozoan of the genus Leishmania, endemic in the Mediterranean basin, are the cause of cutaneous (CL), mucocutaneous (MCL), and visceral leishmaniasis (VL). A 75-year-old woman was admitted nine years after a second kidney transplant (KT), due to persistent pancytopenia and fever. She presented edema and erythema of the nose in the last two years and an exophytic nodular lesion located on the left arm, with areas of peripheral necrosis and central ulceration in the last 18 months. A bone marrow biopsy revealed features compatible with Leishmania amastigotes, and polymerase chain reaction test (PCR) for Leishmania infantum was positive. Moreover, biopsy and PCR for L. infantum of the cutaneous lesion on the patient’s left arm and nose and PCR from peripheral blood were positive. Thus, a diagnosis of CL, MCL, and VL was made, and liposomal amphotericin B was initiated, but the patient had an unfavorable outcome and died. This is the first report of a KT recipient presenting with the entire spectrum of leishmaniasis. In Portugal, this infection is rare—so a high degree of clinical suspicion is required for its diagnosis, especially in endemic regions, as visceral leishmaniasis is a potentially life-threatening infection.

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion-toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 ( TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain.

The optimization of compounds with multiple targets in the drug discovery cycle is a difficult multidimensional problem. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Efficient microwave-assisted synthesis of pteridines along with iterations of crystallographic structure determination were used to validate computational docking predictions and support derivation of a structure-activity relationship for multitarget inhibition. This approach yielded compounds showing picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, along with selective submicromolar inhibition of parasitic dihydrofolate reductase (DHFR). Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei, whilst retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

Canine leishmaniasis is a major veterinary issue and also a public health challenge due to its zoonotic potential. In this context, serological evaluation is essential for Canine leishmaniasis management. Several serological alternatives, such as rapid diagnostic tests, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence antibody test (IFAT), are well established. In fact, the capacity of distinct tests and antigens, evaluated by their sensitivity and specificity, to detect disease is normally considered sufficient for diagnosing Canine leishmaniasis. In this context, we evaluated the seropositivity using 8 different serological tests (ELISA with Leishmania recombinant proteins (rK39, LicTXNPx); soluble promastigote Leishmania antigens (SPLA); commercial ELISA test) in 82 clinically suspect animals from Northern Portugal. The obtained serological data originated 50% of inconclusive serological information with a mixture of seropositive and seronegative results for individual animals. Cut-off independent risk groups were then generated from the serological data to evaluate the clustering of the samples. This analysis originated risk groups that correlated with the most seropositive samples, suggesting that this method might be used, in a cut-off independent manner, to improve conventional serological evaluation. Ultimately, given that no test prioritization exists, the use of any single serological test increases the potential for misdiagnosis, along with all associated risks for the dog as well as public health. The use of a cut-off independent analysis has the potential to improve the predictive values of these tests, enabling a more accurate evaluation of the dog’s condition. Our sincere thanks to Dr. Steven Reed - Infectious Disease Research Institute, Seattle, USA for providing the rK39 used in the study. This work was financed by: FEDER-Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)” Ó. Felgueiras and M. Brito were partially supported by CMUP (UID/MAT/00144/2019), which is funded by FCT (Portugal) with national (MCTES) and European structural funds through the programs FEDER, under the partnership agreement PT2020.

Lutzomyia longipalpis sand flies are the major natural vector of Leishmania infantum parasites, responsible for transmission of visceral leishmaniasis in the New World. Several experimental studies have demonstrated the ability of Lu. longipalpis to sustain development of different Leishmania species. However, no study had explored in depth the potential vector competence of Lu. longipalpis for Leishmania species other than L. infantum. Here, we show that Lu. longipalpis is a competent vector of L. major parasites, being able to acquire parasites from active cutaneous leishmaniasis lesions, sustain mature infections, and transmit them to naive hosts, causing disease.

The LIBRA compound library is a collection of 522 non-commercial molecules contributed by various Italian academic laboratories. These compounds have been designed and synthesized during different medicinal chemistry programs and are hosted by the Italian Institute of Technology. We report the screening of the LIBRA compound library against Trypanosoma brucei and Leishmania major pteridine reductase 1, TbPTR1 and LmPTR1. Nine compounds were active against parasitic PTR1 and were selected for cell-based parasite screening, as single agents and in combination with methotrexate (MTX). The most interesting TbPTR1 inhibitor identified was 4-(benzyloxy)pyrimidine-2,6-diamine (LIB_66). Subsequently, six new LIB_66 derivatives were synthesized to explore its Structure-Activity-Relationship (SAR) and absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The results indicate that PTR1 has a preference to bind inhibitors, which resemble its biopterin/folic acid substrates, such as the 2,4-diaminopyrimidine derivatives.

Leishmaniasis is a spectrum of diseases transmitted by sand fly vectors that deposit Leishmania spp. parasites in the host skin during blood feeding. Currently, available treatment options are limited, associated with high toxicity and emerging resistance. Even though a vaccine for human leishmaniasis is considered an achievable goal, to date we still do not have one available, a consequence (amongst other factors) of a lack of pre-clinical to clinical translatability. Pre-exposure to uninfected sand fly bites or immunization with defined sand fly salivary proteins was shown to negatively impact infection. Still, cross-protection reports are rare and dependent on the phylogenetic proximity of the sand fly species, meaning that the applicability of a sand fly saliva-based vaccine will be limited to a defined geography, one parasite species and one form of leishmaniasis. As a proof of principle of a future vector saliva-based pan-Leishmania vaccine, we engineered through a reverse vaccinology approach that maximizes translation to humans, a fusion protein consisting of immunogenic portions of PdSP15 and LJL143, sand fly salivary proteins demonstrated as potential vaccine candidates against cutaneous and visceral leishmaniasis, respectively. The in silico analysis was validated ex vivo, through T cell proliferation experiments, proving that the fusion protein (administered as a DNA vaccine) maintained the immunogenicity of both PdSP15 and LJL143. Additionally, while no significant effect was detected in the context of L. major transmission by P. duboscqi, this DNA vaccine was defined as partially protective, in the context of L. major transmission by L. longipalpis sand flies. Importantly, a high IFN¿ response alone was not enough to confer protection, that mainly correlated with low T cell mediated Leishmania-specific IL-4 and IL-10 responses, and consequently with high pro/anti-inflammatory cytokine ratios. Overall our immunogenicity data suggests that to design a potentially safe vector-based pan-Leishmania vaccine, without geographic restrictions and against all forms of leishmaniasis is an achievable goal. This is why we propose our approach as a proof-of principle, perhaps not only applicable to the anti-Leishmania vector-based vaccines’ field, but also to other branches of knowledge that require the design of multi-epitope T cell vaccines with a higher potential for translation. The research leading to these results has received funding from the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This research was also supported in part by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. PC was supported by Foundation for Science and Technology (FCT), Portugal, through the individual Grant SFRH/BD/121252/2016. PC was also supported by Fulbright Portugal. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have considered the thiadiazoles opened forms and reaction intermediates, thiosemicarbazones, as compounds of interest for phenotypic screening against Trypanosoma brucei (Tb), intracellular amastigote form of Leishmania infantum (Li) and Trypanosoma cruzi (Tc). Similar compounds have already shown interesting activity against the same organisms. The compounds were particularly effective against T. brucei and T. cruzi. Among the 28 synthesized compounds, the best one was (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene) hydrazinecarbothioamide (A14) yielding a comparable anti-parasitic activity against the three parasitic species (TbEC50 = 2.31 mM, LiEC50 = 6.14 mM, TcEC50 = 1.31 mM) and a Selectivity Index higher than 10 with respect to human macrophages, therefore showing a pan-anti-trypanosomatidic activity. (E)-2-((3'.4'-dimethoxy-[1.1'-biphenyl]-3-yl)methyle ne) hydrazinecarbothioamide (A12) and (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene)hydrazine carbothioamide (A14) were able to potentiate the anti-parasitic activity of methotrexate (MTX) when evaluated in combination against T. brucei, yielding a 6-fold and 4-fold respectively Dose Reduction Index for MTX. The toxicity profile against four human cell lines and a panel of in vitro early-toxicity assays (comprising hERG, Aurora B, five cytochrome P450 isoforms and mitochondrial toxicity) demonstrated the low toxicity for the thosemicarbazones class in comparison with known drugs. The results confirmed thiosemicarbazones as a suitable chemical scaffold with potential for the development of properly decorated new anti-parasitic drugs.

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.

Canine leishmaniosis (CanL) is a major veterinary concern and a public health issue. Serological data are essential for disease management. Several antigens used in serological assays have specificity related problems preventing relevant seropositivity values establishment. Herein we report significant seropositivity level disparity in a study cohort with 384 dogs from eight countries, for antigens traditionally used in CanL - soluble promastigote Leishmania antigens (SPLA) and K39 recombinant protein (rK39): 43·8 and 2·9% for SPLA and rK39, respectively. To better understand the reasons for this disparity, CanL-associated serological response was characterized using, for complement serological evaluation, a ubiquitous antigen - soluble Escherichia coli antigens (SECAs). Using cohorts of CanL dogs and dogs without clinical evidences of CanL from non-endemic regions of Portugal, the serological response of CanL animals followed specific trend of seropositivity rK39 > SPLA > SECA absent in non-diseased animals. Using receiver operating characteristic curve analysis, these characteristic trends were converted in ratios, SPLA/SECA, rK39/SECA and rK39/SPLA, that presented high predictive for discriminating the CanL cohort that was potentiated when applied in a scoring system involving positivity to four out of five predictors (rK39, SPLA, SPLA/SECA, rK39/SECA and rK39/SPLA). In fact, this approach discriminated CanL with similar sensitivity/specificity as reference antigens, diminishing seropositivity in European cohort to 1·8%. Ultimately, non-related antigens like SECA and seropositivity ratios between antigens enable different perspectives into serological data focusing on the search of characteristic serological signatures and not simple absolute serology values contributing to comprehensive serological status characterization. This work was financed by FEDER–Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020–Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project‘Institute for Research and Innovation in Health Sciences’(POCI-01-0145-FEDER-007274) and from the European Community’s Seventh Framework Programme under grant agreement No. 603181 [Clinical Studies on a Multivalent Vaccine for Human Visceral Leishmaniasis (MuLeVaClin)]. ‘This article is a result of the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)’. C.L. and N.S. were supported by BD SFRH/BD/89183/2012 and European Community’s Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED), respectively.

Canine leishmaniosis (CanL) prevention in the Mediterranean basin is considered essential to stop human zoonotic visceral leishmaniasis. In this context, vaccination of dogs is expected to have a significant impact in disease control. CaniLeish® (Virbac Animal Health) is one of a few CanL vaccines that are at this moment licensed in Europe. This vaccine contains purified excreted-secreted proteins of Leishmania having several antigens/immunogens with potential to influence serological response. Therefore, it is important to know if CaniLeish vaccination increased the diagnostic challenges associated with conventional serology, limiting the value of some antigens. To address this 20 dogs from a cohort of 35 healthy dogs that were vaccinated, maintained indoor for 1 month and then returned to their natural domiciles for 2 years. After this period, they were re-called to evaluate their clinical/parasitological condition and assess the evolution of seroreactivity against different antigens: soluble promastigote Leishmania antigens (SPLA), recombinant protein Leishmania infantum cytosolic peroxiredoxin, recombinant protein K39 (rK39), recombinant protein K28 and recombinant kinesin degenerated derived repeat using ELISA. Two years after vaccination all vaccinated non-infected animals were seropositive for SPLA. For the other antigens the serological profile was indistinguishable from non-infected animals. Moreover, vaccinated animals presented a characteristic relative serological profile, with higher normalized serological response to SPLA than rK39. This fact enabled to distinguish with sensitivity 92.3% and specificity 95.4%, vaccinated non-infected dogs from infected and non-infected dogs. Ultimately, relative serological profile enabled the detection of healthy vaccinated animals enabling more accurate serological surveys. This work was financed by: FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project Institute for Research and Innovation in Health Sciences (POCI-01-0145-FEDER-007274) and project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). EC was supported by a research contract funded via the VII PN I+D+I 2013-2016 programme and FEDER Funds (RICET RD12/0018/0003).

Every year, up to 90,000 new cases of Visceral Leishmaniasis and 30,000 resultant deaths are estimated to occur worldwide. Such numbers give relevance to the continuous study of this complex form of the disease: a zoonosis and an anthroponosis; two known etiological agents (Leishmania infantum and L. donovani, respectively); with an estimated average ratio of 1 symptomatic per 10 asymptomatic individuals; and sometimes associated with atypical clinical presentations. This complexity, which results from a long co-evolutionary process involving vector-host, host-pathogen, and pathogen-vector interactions, is still not completely understood. The determinants of visceralization are not fully defined and the dichotomy resistance vs. susceptibility remains unsolved, translating into obstacles that delay the progress of global disease control. Inbred mouse models, with different susceptibility patterns to Leishmania infection, have been very useful in exploring this dichotomy. BALB/c and C57BL/6 mice were described as susceptible strains to L. donovani visceral infection, while SV/129 was considered resistant. Here, we used these three mouse models, but in the context of L. infantum infection, the other Leishmania species that cause visceral disease in humans, and dynamically compared their local and systemic infection-induced immune responses in order to establish a parallel and to ultimately better understand susceptibility vs. resistance in visceral leishmaniasis. Overall, our results suggest that C57BL/6 mice develop an intermediate "infection-phenotype" in comparison to BALB/c and SV/129 mouse strains, considering both the splenic parasite burden and the determined target organs weights. However, the immune mechanisms associated with the control of infection seem to be different in each mouse strain. We observed that both BALB/c and SV/129, but not C57BL/6 mice, show an infection-induced increase of splenic T follicular helper cells. On the other hand, differences detected in terms of CD21 expression by B cells early after infection, together with the quantified anti-Leishmania specific antibodies, suggest that SV/129 are faster than BALB/c and C57BL/6 mice in the assembly of an efficient B-cell response. Additionally, we observed an infection-induced increase in polyfunctional CD4+ T cells in the resistant SV/129 model, opposing an infection-induced increase in CD4+IL-10+ cells in susceptible BALB/c mice. Our data aligns with the observations reported for L. donovani infection and suggest that not only a single mechanism, but an interaction of several could be necessary for the control of this parasitic disease. The research leading to these results has received funding from the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). PC was supported by Foundation for Science and Technology (FCT), Portugal, through the individual grant SFRH/BD/121252/2016.