Potential selection of antimony and methotrexate cross-resistance in Leishmania infantum circulating strains.

BACKGROUND: Visceral leishmaniasis (VL) resolution depends on a wide range of factors, including the instauration of an effective treatment coupled to a functional host immune system. Patients with a depressed immune system, like the ones receiving methotrexate (MTX), are at higher risk of developing VL and refusing antileishmanial drugs. Moreover, the alarmingly growing levels of antimicrobial resistance, especially in endemic areas, contribute to the increasing the burden of this complex zoonotic disease. PRINCIPAL FINDINGS: To understand the potential links between immunosuppressants and antileishmanial drugs, we have studied the interaction of antimony (Sb) and MTX in a Leishmania infantum reference strain (LiWT) and in two L. infantum clinical strains (LiFS-A and LiFS-B) naturally circulating in non-treated VL dogs in Spain. The LiFS-A strain was isolated before Sb treatment in a case that responded positively to the treatment, while the LiFS-B strain was recovered from a dog before Sb treatment, with the dog later relapsing after the treatment. Our results show that, exposure to Sb or MTX leads to an increase in the production of reactive oxygen species (ROS) in LiWT which correlates with a sensitive phenotype against both drugs in promastigotes and intracellular amastigotes. LiFS-A was sensitive against Sb but resistant against MTX, displaying high levels of protection against ROS when exposed to MTX. LiFS-B was resistant to both drugs. Evaluation of the melting proteomes of the two LiFS, in the presence and absence of Sb and MTX, showed a differential enrichment of direct and indirect targets for both drugs, including common and unique pathways. CONCLUSION: Our results show the potential selection of Sb-MTX cross-resistant parasites in the field, pointing to the possibility to undermine antileishmanial treatment of those patients being treated with immunosuppressant drugs in Leishmania endemic areas.

Exploring direct and indirect targets of current antileishmanial drugs using a novel thermal proteomics profiling approach.

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an oft-fatal neglected tropical disease. In the absence of an effective vaccine, the control of leishmaniasis relies exclusively on chemotherapy. Due to the lack of established molecular/genetic markers denoting parasite resistance, clinical treatment failure is often used as an indicator. Antimony-based drugs have been the standard antileishmanial treatment for more than seven decades, leading to major drug resistance in certain regions. Likewise, drug resistance to miltefosine and amphotericin B continues to spread at alarming rates. In consequence, innovative approaches are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. To this end, we have implemented a novel approach based on thermal proteome profiling (TPP) to further characterize the mode of action of antileishmanials antimony, miltefosine and amphotericin B, as well as to better understand the mechanisms of drug resistance deployed by Leishmania. Proteins become more resistant to heat-induced denaturation when complexed with a ligand. In this way, we used multiplexed quantitative mass spectrometry-based proteomics to monitor the melting profile of thousands of expressed soluble proteins in WT, antimony-resistant, miltefosine-resistant, and amphotericin B-resistant L. infantum parasites, in the presence (or absence) of the above-mentioned drugs. Bioinformatics analyses were performed, including data normalization, melting profile fitting, and identification of proteins that underwent changes (fold change > 4) caused by complexation with a drug. With this unique approach, we were able to narrow down the regions of the L. infantum proteome that interact with antimony, miltefosine, and amphotericin B; validating previously-identified and unveiling novel drug targets. Moreover, analyses revealed candidate proteins potentially involved in drug resistance. Interestingly, we detected thermal proximity coaggregation for several proteins belonging to the same metabolic pathway (i.e., tryparedoxin peroxidase and aspartate aminotransferase in proteins exposed to antimony), highlighting the importance of these pathways. Collectively, our results could serve as a jumping-off point for the future development of innovative diagnostic tools for the detection and evaluation of antimicrobial-resistant Leishmania populations, as well as open the door for new on-target therapies.

Identification of asymptomatic Leishmania infections: a scoping review.

BACKGROUND: Asymptomatic Leishmania infection may play an important role in the transmission of the parasite in endemic areas. At present there is no consensus on the definition of asymptomatic Leishmania infection, nor is there a safe and accessible gold standard test for its identification. METHODS: This paper presents a scoping review to summarize definitions of asymptomatic Leishmania infection found in the literature, as well as to detail the approach (molecular, serological, cellular, and/or parasitological tests) used by researchers to identify this asymptomatic population. A scoping review of published and gray literature related to asymptomatic Leishmania infection was conducted; retrieved citations were screened based on predefined eligibility criteria, and relevant data items were extracted from eligible articles. The analysis is descriptive and is presented using tables, figures, and thematic narrative synthesis. RESULTS: We conducted a screening of 3008 articles, of which 175 were selected for the full review. Of these articles, we selected 106 that met the inclusion criteria. These articles were published between 1991 and 2021, and in the last 5 years, up to 38 articles were reported. Most of the studies were conducted in Brazil (26%), Spain (14%), India (12%), Bangladesh (10%), and Ethiopia (7%). Of the studies, 84.9% were conducted in the immunocompetent population, while 15.1% were conducted in the immunosuppressed population (HIV, immunosuppressive drugs, and organ transplantation population). We report 14 different techniques and 10 strategies employed by researchers to define asymptomatic Leishmania infection in an endemic area. CONCLUSIONS: The definition of asymptomatic Leishmania infection is not unified across the literature, but often includes the following criteria: residence (or extended stay) in a Leishmania-endemic area, no reported signs/symptoms compatible with leishmaniasis, and positive on a combination of serological, molecular, cellular, and/or parasitological tests. Caution is recommended when comparing results of different studies on the subject of asymptomatic infections, as the reported prevalence cannot be confidently compared between areas due to the wide variety of tests employed by research groups. More research on the importance of asymptomatic immunosuppressed and immunocompetent Leishmania-positive populations in leishmaniasis epidemiology is required.

Three different mutations in the DNA topoisomerase 1B in Leishmania infantum contribute to resistance to antitumor drug topotecan.

BACKGROUND: The evolution of drug resistance is one of the biggest challenges in leishmaniasis and has prompted the need for new antileishmanial drugs. Repurposing of approved drugs is a faster and very attractive strategy that is gaining supporters worldwide. Different anticancer topoisomerase 1B (TOP1B) inhibitors have shown strong antileishmanial activity and promising selective indices, supporting the potential repurposing of these drugs. However, cancer cells and Leishmania share the ability to become rapidly resistant. The aim of this study was to complete a whole-genome exploration of the effects caused by exposure to topotecan in order to highlight the potential mechanisms deployed by Leishmania to favor its survival in the presence of a TOP1B inhibitor. METHODS: We used a combination of stepwise drug resistance selection, whole-genome sequencing, functional validation, and theoretical approaches to explore the propensity of and potential mechanisms deployed by three independent clones of L. infantum to resist the action of TOP1B inhibitor topotecan. RESULTS: We demonstrated that L. infantum is capable of becoming resistant to high concentrations of topotecan without impaired growth ability. No gene deletions or amplifications were identified from the next-generation sequencing data in any of the three resistant lines, ruling out the overexpression of efflux pumps as the preferred mechanism of topotecan resistance. We identified three different mutations in the large subunit of the leishmanial TOP1B (Top1BF187Y, Top1BG191A, and Top1BW232R). Overexpression of these mutated alleles in the wild-type background led to high levels of resistance to topotecan. Computational molecular dynamics simulations, in both covalent and non-covalent complexes, showed that these mutations have an effect on the arrangement of the catalytic pentad and on the interaction of these residues with surrounding amino acids and DNA. This altered architecture of the binding pocket results in decreased persistence of topotecan in the ternary complex. CONCLUSIONS: This work helps elucidate the previously unclear potential mechanisms of topotecan resistance in Leishmania by mutations in the large subunit of TOP1B and provides a valuable clue for the design of improved inhibitors to combat resistance in both leishmaniasis and cancer. Our data highlights the importance of including drug resistance evaluation in drug discovery cascades.

Influence of N-Methylation and Conformation on Almiramide Anti-Leishmanial Activity.

The almiramide N-methylated lipopeptides exhibit promising activity against trypanosomatid parasites. A structure-activity relationship study has been performed to examine the influences of N-methylation and conformation on activity against various strains of leishmaniasis protozoan and on cytotoxicity. The synthesis and biological analysis of twenty-five analogs demonstrated that derivatives with a single methyl group on either the first or fifth residue amide nitrogen exhibited greater activity than the permethylated peptides and relatively high potency against resistant strains. Replacement of amino amide residues in the peptide, by turn inducing α amino γ lactam (Agl) and N-aminoimidazalone (Nai) counterparts, reduced typically anti-parasitic activity; however, peptide amides possessing Agl residues at the second residue retained significant potency in the unmethylated and permethylated series. Systematic study of the effects of methylation and turn geometry on anti-parasitic activity indicated the relevance of an extended conformer about the central residues, and conformational mobility by tertiary amide isomerization and turn geometry at the extremities of the active peptides.

Of Drugs and Trypanosomatids: New Tools and Knowledge to Reduce Bottlenecks in Drug Discovery.

Leishmaniasis (Leishmania species), sleeping sickness (Trypanosoma brucei), and Chagas disease (Trypanosoma cruzi) are devastating and globally spread diseases caused by trypanosomatid parasites. At present, drugs for treating trypanosomatid diseases are far from ideal due to host toxicity, elevated cost, limited access, and increasing rates of drug resistance. Technological advances in parasitology, chemistry, and genomics have unlocked new possibilities for novel drug concepts and compound screening technologies that were previously inaccessible. In this perspective, we discuss current models used in drug-discovery cascades targeting trypanosomatids (from in vitro to in vivo approaches), their use and limitations in a biological context, as well as different examples of recently discovered lead compounds.