Gene disruption of the DNA topoisomerase IB small subunit induces a non-viable phenotype in the hemoflagellate Leishmania major

<p>Abstract</p> <p>Background</p> <p>The unusual heterodimeric leishmanial DNA topoisomerase IB consists of a large subunit containing the phylogenetically conserved "core" domain, and a small subunit harboring the C-terminal region with the characteristic tyrosine residue in the active site. RNAi silencing of any of both protomers induces a non-viable phenotype in the hemoflagelate <it>Trypanosoma brucei</it>. Unfortunately, this approach is not suitable in <it>Leishmania </it>where gene replacement with an antibiotic marker is the only approach to generate lack-of-function mutants. In this work, we have successfully generated null mutants in the small subunit of the <it>L. major </it>DNA topoisomerase IB using two selection markers, each conferring resistance to hygromycin B and puromycin, respectively.</p> <p>Results</p> <p>We have successfully replaced both <it>topS </it>loci with two selection markers. However, to achieve the second transfection round, we have had to rescue the null-homozygous with an episomal vector carrying the <it>Leishmania major topS </it>gene. Phenotypic characterization of the <it>L. major </it>rescued strain and a <it>L. major </it>strain, which co-overexpresses both subunits, shows few differences in DNA relaxation and camptothecin cytotoxicity when it was compared to the wild-type strain. Studies on phosphatidylserine externalization show a poor incidence of camptothecin-induced programmed cell death in <it>L. major</it>, but an effective cell-cycle arrest occurs within the first 24 h. S-Phase delay and G₂/M reversible arrest was the main outcome at lower concentrations, but irreversible G₂arrest was detected at higher camptothecin pressure.</p> <p>Conclusion</p> <p>Results obtained in this work evidence the essentiality of the <it>topS </it>gene encoding the <it>L. major </it>DNA topoisomerase IB small subunit. Reversibility of the camptothecin effect points to the existence of effective checkpoint mechanisms in <it>Leishmania </it>parasites.</p

Miltefosine and nifuratel combination: a promising therapy for the treatment of leishmania donovani visceral leishmaniasis

[EN] Visceral leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania donovani and Leishmania infantum that is endemic not only in East African countries, but also in Asia, regions of South America and the Mediterranean Basin. For the pharmacological control of this disease, there is a limited number of old and, in general, poorly adherent drugs, with a multitude of adverse effects and low oral bioavailability, which favor the emergence of resistant pathogens. Pentavalent antimonials are the first-line drugs, but due to their misuse, resistant Leishmania strains have emerged worldwide. Although these drugs have saved many lives, it is recommended to reduce their use as much as possible and replace them with novel and more friendly drugs. From a commercial collection of anti-infective drugs, we have recently identified nifuratel-a nitrofurantoin used against vaginal infections-as a promising repurposing drug against a mouse model of visceral leishmaniasis. In the present work, we have tested combinations of miltefosine-the only oral drug currently used against leishmaniasis-with nifuratel in different proportions, both in axenic amastigotes from bone marrow and in intracellular amastigotes from infected Balb/c mouse spleen macrophages, finding a potent synergy in both cases. In vivo evaluation of oral miltefosine/nifuratel combinations using a bioimaging platform has revealed the potential of these combinations for the treatment of this disease.SIThis research was partially funded by MINECO; SAF2017-83575-R and PID 2020-119031RB-100 to R.M.R. E.M.-F. is contracted with EU PRIMA (PCI2022-132925); G.G. is recipient of FPI scholarship (PRE 2021 096909) supported by AE

Phenotype Screening of an Azole-bisindole Chemical Library Identifies URB1483 as a New Antileishmanial Agent Devoid of Toxicity on Human Cells

none11sìWe report the evaluation of a small library of azole-bisindoles for their antileishmanial potential, in terms of efficacy on Leishmania infantum promastigotes and intracellular amastigotes. Nine compounds showed good activity on L. infantum MHOM/TN/80/IPT1 promastigotes with IC50 values ranging from 4 to 10 μM. These active compounds were also tested on human (THP-1, HEPG2, HaCaT, and human primary fibroblasts) and canine (DH82) cell lines. URB1483 was selected as the best compound, with no quantifiable cytotoxicity in mammalian cells, to test the efficacy on intracellular amastigotes. URB1483 significantly reduced the infection index of both human and canine macrophages with an effect comparable to the clinically used drug pentamidine. URB1483 emerges as a new anti-infective agent with remarkable antileishmanial activity and no cytotoxic effects on human and canine cells.openDiotallevi, Aurora; Scalvini, Laura; Buffi, Gloria; Pérez-Pertejo, Yolanda; De Santi, Mauro; Verboni, Michele; Favi, Gianfranco; Magnani, Mauro; Lodola, Alessio; Lucarini, Simone; Galluzzi, LucaDiotallevi, Aurora; Scalvini, Laura; Buffi, Gloria; Pérez-Pertejo, Yolanda; De Santi, Mauro; Verboni, Michele; Favi, Gianfranco; Magnani, Mauro; Lodola, Alessio; Lucarini, Simone; Galluzzi, Luc

Hybrid Quinolinyl Phosphonates as Heterocyclic Carboxylate Isosteres: Synthesis and Biological Evaluation against Topoisomerase 1B (TOP1B)

This work describes, for the first time, the synthesis of dialkyl (2-arylquinolin-8-yl)phosphonate derivatives. The preparation was carried out through a direct and simple process as a multicomponent Povarov reaction of aminophenylphosphonates, aldehydes, and styrenes and subsequent oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or, alternatively, by a cycloaddition reaction between phosphonate aldimines and acetylenes. Based on phosphonate group structural characteristics, considered as phosphorous isosteres of carboxylic heterocycles, they may present interesting biological properties related to cell proliferation. In the current report, a new series of dialkyl (2-arylquinolin-8-yl)phosphonates have been synthesized and their antiproliferative effect evaluated on different human cancer and embryonic cells, as well as on Leishmania infantum parasites, a eukaryotic protist responsible for visceral leishmaniasis. Thereby, the antitumor effect was assessed in human lung adenocarcinoma cells (A549), human ovarian carcinoma cells (SKOV3), and human embryonic kidney cells (HEK293) versus the non-cancerous lung fibroblasts cell line (MRC5). On the other hand, the antileishmanial activity was tested against both stages of L. infantum cell cycle, namely free-living promastigotes and intramacrophage amastigotes, using a primary culture of Balb/c splenocytes to calculate the selectivity index. Besides the antiproliferative and antileishmanial capacities, their behavior as topoisomerase 1B inhibitors has been evaluated as a possible mechanism of action.This research was financially supported by Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) y Fondo Europeo de Desarrollo Regional (FEDER; RTI2018–101818-B-I00, UE) and by Gobierno Vasco, Universidad del País Vasco (GV, IT 992–16; UPV) is gratefully acknowledged. Technical and human support provided by IZO-SGI, SGIker (UPV/EHU, MICINN, GV/EJ, ERDF, and ESF) is gratefully acknowledged. AS thanks the Basque Government for a formation contract. This collaborative research was funded by MINECO; SAF2017–83575-R to RMR

Axenic interspecies and intraclonal hybrid formation in Leishmania: Successful crossings between visceral and cutaneous strains

[EN] Neglected Tropical Diseases (NTDs) represent a serious threat to humans, especially for those living in poor or developing countries. Leishmanianiosis is considered a zoonotic NTD transmitted by the bite of female phlebotomine sandflies, and is manifested mainly as a visceral form (caused by L. infantum and L. donovani) and a cutaneous form (caused by many species including L. major, L. tropica and L. braziliensis). Although it is now known that sexual reproduction occurs in these parasites, more studies are necessary to characterize the ability of Leishmania to generate hybrids, which may represent an important mechanism for virulence, drug resistance or adaptation to the host immune system. Therefore, several experiments were conducted to generate either intraclonal or interspecies hybrids in vitro. Results demonstrated that hybrids can be formed even with outcrosses between parasites causing visceral and cutaneous forms of the disease. Characterization of hybrids in terms of ploidy, kDNA content, growth rate and infection capacity provide important information about sexual reproduction in these parasites.SI: C.G.C (LE255-16) and B.D.A (LE208-17) are recipients of Junta de Castilla y Leo´n (JCyL) and European Social Found (ESF)’s Fellowships Scheme for Doctoral Training Programs. This research was funded by MINECO; SAF2017- 83575-R to RMR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Antiparasitic effect of synthetic aromathecins on Leishmania infantum

[EN} Background Canine leishmaniasis is a zoonotic disease caused by Leishmania infantum, being the dogs one of the major reservoirs of human visceral leishmaniasis. DNA topology is a consolidated target for drug discovery. In this regard, topoisomerase IB – one of the enzymes controlling DNA topology – has been poisoned by hundreds of compounds that increase DNA fragility and cell death. Aromathecins are novel molecules with a multiheterocyclic ring scaffold that have higher stability than camptothecins. Results Aromathecins showed strong activity against both forms of L. infantum parasites, free-living promastigotes and intra-macrophagic amastigotes harbored in ex vivo splenic explant cultures obtained from infected BALB/c mice. However, they prevented the relaxation activity of leishmanial topoisomerase IB weakly, which suggests that the inhibition of topoisomerase IB partially explains the antileishmanial effect of these compounds. The effect of aromathecins was also studied against a strain resistant to camptothecin, and results suggested that the trafficking of these compounds is not through the ABCG6 transporter. Conclusions Aromathecins are promising novel compounds against canine leishmaniasis that can circumvent potential resistances based on drug efflux pumps.SIThis research had the financial support of: Ministerio de Economía y Competitividad (MINECO, AEI, FEDER, UE) [MINECO: AGL2016–79813-C2-1R, SAF2017–83575-R]. Junta de Castilla y León cofinanced by FEDER UE [LE020P17, Grant UIC108]. The funding body does not participate in the design of the study; collection, analysis and interpretation of data and in writing the manuscript

Antileishmanial activity of terpenylquinones on Leishmania infantum and their effects on Leishmania topoisomerase IB

[EN] Leishmania is the aethiological agent responsible for the visceral leishmaniasis, a serious parasite-borne disease widely spread all over the World. The emergence of resistant strains makes classical treatments less effective; therefore, new and better drugs are necessary. Naphthoquinones are interesting compounds for which many pharmacological properties have been described, including leishmanicidal activity. This work shows the antileishmanial effect of two series of terpenyl-1,4-naphthoquinones (NQ) and 1,4-anthraquinones (AQ) obtained from natural terpenoids, such as myrcene and myrceocommunic acid. They were evaluated both in vitro and ex vivo against the transgenic iRFP-Leishmania infantum strain and also tested on liver HepG2 cells to determine their selectivity indexes. The results indicated that NQ derivatives showed better antileishmanial activity than AQ analogues, and among them, compounds with a diacetylated hydroquinone moiety provided better results than their corresponding quinones. Regarding the terpenic precursor, compounds obtained from the monoterpenoid myrcene displayed good antiparasitic efficiency and low cytotoxicity for mammalian cells, whereas those derived from the diterpenoid showed better antileishmanial activity without selectivity. In order to explore their mechanism of action, all the compounds have been tested as potential inhibitors of Leishmania type IB DNA topoisomerases, but only some compounds that displayed the quinone ring were able to inhibit the recombinant enzyme in vitro. This fact together with the docking studies performed on LTopIB suggested the existence of another mechanism of action, alternative or complementary to LTopIB inhibition. In silico druglikeness and ADME evaluation of the best leishmanicidal compounds has shown good predictable druggabilitySIFinancial support came from Spanish MINECO (CTQ2015-68175-R, AGL2016-79813-C2-1-R, AGL2016-79813-C2-2-R and SAF2017-83575-R), ISCIII-RICET Network (RD12/0018/0002) and Consejería de Educación de la Junta de Castilla y León (LE020P17) co-financed by the Fondo Social Europeo of the European Union (FEDER-EU). P. G. J. acknowledges funding by Fundación Salamanca Ciudad de Cultura y Saberes (’‘Programme for attracting scientific talent to Salamanca’‘

Gel-Free Tools for Quick and Simple Screening of Anti-Topoisomerase 1 Compounds

With the increasing need for effective compounds against cancer or pathogen-borne diseases, the development of new tools to investigate the enzymatic activity of biomarkers is necessary. Among these biomarkers are DNA topoisomerases, which are key enzymes that modify DNA and regulate DNA topology during cellular processes. Over the years, libraries of natural and synthetic small-molecule compounds have been extensively investigated as potential anti-cancer, anti-bacterial, or anti-parasitic drugs targeting topoisomerases. However, the current tools for measuring the potential inhibition of topoisomerase activity are time consuming and not easily adaptable outside specialized laboratories. Here, we present rolling circle amplification-based methods that provide fast and easy readouts for screening of compounds against type 1 topoisomerases. Specific assays for the investigation of the potential inhibition of eukaryotic, viral, or bacterial type 1 topoisomerase activity were developed, using human topoisomerase 1, Leishmania donovani topoisomerase 1, monkeypox virus topoisomerase 1, and Mycobacterium smegmatis topoisomerase 1 as model enzymes. The presented tools proved to be sensitive and directly quantitative, paving the way for new diagnostic and drug screening protocols in research and clinical settings.This research was funded in part by the Ministerio de Ciencia e Innovación, Spain (PID2021-122558OB-I00, UE) and by Gobierno Vasco, Universidad del País Vasco (GV, IT1701-22; UPV)

Simple and Fast DNA Based Sensor System for Screening of Small-Molecule Compounds Targeting Eukaryotic Topoisomerase 1

Background: Eukaryotic topoisomerase 1 is a potential target of anti-parasitic and anti-cancer drugs. Parasites require topoisomerase 1 activity for survival and, consequently, compounds that inhibit topoisomerase 1 activity may be of interest. All effective topoisomerase 1 drugs with anti-cancer activity act by inhibiting the ligation reaction of the enzyme. Screening for topoisomerase 1 targeting drugs, therefore, should involve the possibility of dissecting which step of topoisomerase 1 activity is affected. Methods: Here we present a novel DNA-based assay that allows for screening of the effect of small-molecule compounds targeting the binding/cleavage or the ligation steps of topoisomerase 1 catalysis. This novel assay is based on the detection of a rolling circle amplification product generated from a DNA circle resulting from topoisomerase 1 activity. Results: We show that the binding/cleavage and ligation reactions of topoisomerase 1 can be investigated separately in the presented assay termed REEAD (C|L) and demonstrate that the assay can be used to investigate, which of the individual steps of topoisomerase 1 catalysis are affected by small-molecule compounds. The assay is gel-free and the results can be detected by a simple colorimetric readout method using silver-on-gold precipitation rendering large equipment unnecessary. Conclusion: REEAD (C|L) allows for easy and quantitative investigations of topoisomerase 1 targeting compounds and can be performed in non-specialized laboratories.K.V. thanks Aase and Ejnar Danielsens Fond, Aage and Johanne Louis-Hansens Fond and Edel and Wilhelm Daubenmerkls Almenvelgørende Fond; A.S. thanks the Basque Government for a formation contract

Deletion Study of DNA Topoisomerase IB from Leishmania donovani: Searching for a Minimal Functional Heterodimer

The substantial differences between trypanosomal and leishmanial DNA topoisomerase IB concerning to their homologues in mammals have provided a new lead in the study of the structural determinants that can be effectively targeted. Leishmania donovani, the causative agent of visceral leishmaniasis, contains an unusual heterodimeric DNA topoisomerase IB. The catalytically active enzyme consists of a large subunit (LdTopIL), which contains the non-conserved N-terminal end and the phylogenetically conserved “core” domain, and of a small subunit (LdTopIS) which harbors the C-terminal region with the characteristic tyrosine residue in the active site. Heterologous co-expression of LdTopIL and LdTopIS genes in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme LdTopIL/S which can be used for structural studies. An approach by combinatorial cloning of deleted genes encoding for truncated versions of both subunits was used in order to find out structural insights involved in enzyme activity or protein-protein interaction. The role played by the non-conserved N-terminal extension of LdTopIL in both relaxation activity and CPT sensitivity has been examined co-expressing the full-length LdTopIS and a fully active LdTopIΔS deletion with several deletions of LdTopIL lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 26 amino acids placed at the N-terminal end and a variable region comprised between Ala548 to end of the C-terminal extension of LdTopIL were enzymatically dispensable. Altogether this combinatorial approach provides important structural insights of the regions involved in relaxation activity and for understanding the atypical structure of this heterodimeric enzyme