Acanthamoeba spp. can cause a sight threatening disease. At present, the current treatments used to treat Acanthamoeba spp. Infections, such as biguanide-based antimicrobials, remain inefficacious, with the appearance of resistant forms and high cytotoxicity to host cells. In this study, an initial screening was conducted against Acanthamoeba castellanii Neff and murine macrophages J774A.1 using alamarBlue™. Among the 160 compounds included in the cited box, 90% exhibited an inhibition of the parasite above 80%, while only 18.75% of the compounds inhibited the parasite with a lethality towards murine macrophage lower than 20%. Based on the amoebicidal activity, the cytotoxicity assay, and availability, Terconazole was chosen for the elucidation of the action mode in two clinical strains, Acanthamoeba culbertsoni and Acanthamoeba castellanii L10. A fluorescence image-based system and proteomic techniques were used to investigate the effect of the present azole on the cytoskeleton network and various programmed cell death features, including chromatin condensation and mitochondria dysfunction. Taking all the results together, we can suggest that Terconazole can induce programmed cell death (PCD) via the inhibition of sterol biosynthesis inhibition.

Consorcio Centro de Investigación on Biomédica En Red Instituto de Salud Carlos 3 28220 Madrid Spain

Departamento de Obstetricia y Ginecología Pediatría Medicina Preventiva y Salud Pública Toxicología Medicina Legal y Forense y Parasitología Universidad de La Laguna 38200 San Cristóbal de La Laguna Spain

Department of Parasitology Faculty of Science Charles University BIOCEV 252 50 Vestec Czech Republic

Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias Avda Astrofísico Fco Sánchez S N 38200 San Cristóbal de La Laguna Spain

Zobrazit více v PubMed

Reyes-Batlle M., Córdoba-Lanús E., Domínguez-de-Barros A., Sifaoui I., Rodríguez-Expósito R.L., Mantesa-Rodríguez S., Piñero J.E., Lorenzo-Morales J. Reliable and specific detection of Acanthamoeba spp. in dishcloths using quantitative real-time PCR assay. Food Microbiol. 2024;122:104562. doi: 10.1016/j.fm.2024.104562. PubMed DOI

Siddiqui R., Khan N.A. Biology and pathogenesis of Acanthamoeba. Parasites Vectors. 2012;5:6. doi: 10.1186/1756-3305-5-6. PubMed DOI PMC

Wang Y., Jiang L., Zhao Y., Ju X., Wang L., Jin L., Fine R.D., Li M. Biological characteristics and pathogenicity of Acanthamoeba. Front. Microbiol. 2023;14:1147077. doi: 10.3389/fmicb.2023.1147077. PubMed DOI PMC

Fanselow N., Sirajuddin N., Yin X., Huang A.J.W., Stuart P.M. Acanthamoeba Keratitis, Pathology, Diagnosis and Treatment. Pathogens. 2021;10:323. doi: 10.3390/pathogens10030323. PubMed DOI PMC

Sifaoui I., Reyes-Batlle M., López-Arencibia A., Chiboub O., Bethencourt-Estrella C.J., San Nicolás-Hernández D., Rodríguez Expósito R.L., Rizo-Liendo A., Piñero J.E., Lorenzo-Morales J. Screening of the pathogen box for the identification of anti-Acanthamoeba agents. Exp. Parasitol. 2019;201:90–92. doi: 10.1016/j.exppara.2019.04.013. PubMed DOI

Botella L.M. Drug repurposing as a current strategy in medicine discovery. Semer. Med. Fam. 2022;48:101790. doi: 10.1016/j.semerg.2022.03.003. PubMed DOI

Barnes C.B.G., Dans M.G., Jonsdottir T.K., Crabb B.S., Gilson P.R. PfATP4 inhibitors in the Medicines for Malaria Venture Malaria Box and Pathogen Box block the schizont-to-ring transition by inhibiting egress rather than invasion. Front. Cell. Infect. Microbiol. 2022;12:1060202. doi: 10.3389/fcimb.2022.1060202. PubMed DOI PMC

Samby K., Willis P.A., Burrows J.N., Laleu B., Webborn P.J.H. Actives from MMV Open Access Boxes? A suggested way forward. PLoS Pathog. 2021;17:e1009384. doi: 10.1371/journal.ppat.1009384. PubMed DOI PMC

Lopez-Arencibia A., Sifaoui I., Reyes-Batlle M., Bethencourt-Estrella C.J., San Nicolas-Hernandez D., Lorenzo-Morales J., Pinero J.E. Discovery of New Chemical Tools against Leishmania amazonensis via the MMV Pathogen Box. Pharmaceuticals. 2021;14:1219. doi: 10.3390/ph14121219. PubMed DOI PMC

Duffy S., Sykes M.L., Jones A.J., Shelper T.B., Simpson M., Lang R., Poulsen S., Sleebs B.E., Avery V.M. Screening the Medicines for Malaria Venture Pathogen Box across Multiple Pathogens Reclassifies Starting Points for Open-Source Drug Discovery. Antimicrob. Agents Chemother. 2017;61:e00379-17. doi: 10.1128/AAC.00379-17. PubMed DOI PMC

Spalenka J., Escotte-Binet S., Bakiri A., Hubert J., Renault J., Velard F., Duchateau S., Aubert D., Huguenin A., Villena I. Discovery of New Inhibitors of Toxoplasma gondii via the Pathogen Box. Antimicrob. Agents Chemother. 2018;62:e01640-17. doi: 10.1128/AAC.01640-17. PubMed DOI PMC

Dos Santos B.R., Ramos A.B.d.S.B., de Menezes R.P.B., Scotti M.T., Colombo F.A., Marques M.J., Reimao J.Q. Repurposing the Medicines for Malaria Venture’s COVID Box to discover potent inhibitors of Toxoplasma gondii, and in vivo efficacy evaluation of almitrine bismesylate (MMV1804175) in chronically infected mice. PLoS ONE. 2023;18:e0288335. doi: 10.1371/journal.pone.0288335. PubMed DOI PMC

Chao-Pellicer J., Arberas-Jiménez I., Sifaoui I., Piñero J.E., Lorenzo-Morales J. Exploring therapeutic approaches against Naegleria fowleri infections through the COVID box. Int. J. Parasitol. Drugs Drug Resist. 2024;25:100545. doi: 10.1016/j.ijpddr.2024.100545. PubMed DOI PMC

Sivandzade F., Bhalerao A., Cucullo L. Analysis of the Mitochondrial Membrane Potential Using the Cationic JC-1 Dye as a Sensitive Fluorescent Probe. Bio Protoc. 2019;9:e3128. doi: 10.21769/BioProtoc.3128. PubMed DOI PMC

Hua Y., Dai X., Xu Y., Xing G., Liu H., Lu T., Chen Y., Zhang Y. Drug repositioning: Progress and challenges in drug discovery for various diseases. Eur. J. Med. Chem. 2022;234:114239. doi: 10.1016/j.ejmech.2022.114239. PubMed DOI PMC

Pareek S., Huang Y., Nath A., Huang R.S. Chapter 6—The success story of drug repurposing in breast cancer. In: To K.K.W., Cho W.C.S., editors. Drug Repurposing in Cancer Therapy. Academic Press; Cambridge, MA, USA: 2020. pp. 173–190.

Teixeira M.M., Carvalho D.T., Sousa E., Pinto E. New Antifungal Agents with Azole Moieties. Pharmaceuticals. 2022;15:1427. doi: 10.3390/ph15111427. PubMed DOI PMC

Henriquez F.L., Ingram P.R., Muench S.P., Rice D.W., Roberts C.W. Molecular Basis for Resistance of Acanthamoeba Tubulins to All Major Classes of Antitubulin Compounds. Antimicrob. Agents Chemother. 2007;52:1133. doi: 10.1128/aac.00355-07. PubMed DOI PMC

Shing B., Balen M., Mckerrow J.H., Debnath A. Acanthamoeba Keratitis: An update on amebicidal and cysticidal drug screening methodologies and potential treatment with azole drugs. Expert Rev. Anti Infect. Ther. 2022;19:1427. doi: 10.1080/14787210.2021.1924673. PubMed DOI PMC

Bahy R., Helal D. Evaluation of the Antimycotic activity of Terconazole proniosomal Gel. Egypt. J. Med. Microbiol. 2022;31:121–126. doi: 10.21608/ejmm.2022.229668. DOI

Lee J.S., Oh Y., Park J.H., Kyung S.Y., Kim H.S., Yoon S. Terconazole, an Azole Antifungal Drug, Increases Cytotoxicity in Antimitotic Drug-Treated Resistant Cancer Cells with Substrate-Specific P-gp Inhibitory Activity. Int. J. Mol. Sci. 2022;23:13809. doi: 10.3390/ijms232213809. PubMed DOI PMC

Reigada C., Saye M., Valera-Vera E., Miranda M.R., Pereira C.A. Repurposing of terconazole as an anti Trypanosoma cruzi agent. Heliyon. 2019;5:e01947. doi: 10.1016/j.heliyon.2019.e01947. PubMed DOI PMC

Yang S., Yan D., Li M., Li D., Zhang S., Fan G., Peng L., Pan S. Ergosterol depletion under bifonazole treatment induces cell membrane damage and triggers a ROS-mediated mitochondrial apoptosis in Penicillium expansum. Fungal Biol. 2022;126:1–10. doi: 10.1016/j.funbio.2021.09.002. PubMed DOI

Pratiwi R.A., Yahya N.S.W., Chi Y. Bio function of Cytochrome P450 on fungus: A review. IOP Conf. Ser. Earth Environ. Sci. 2022;959:12023. doi: 10.1088/1755-1315/959/1/012023. DOI

Yoshida Y. Cytochrome P450 of fungi: Primary target for azole antifungal agents. Curr. Top. Med. Mycol. 1988;2:388–418. doi: 10.1007/978-1-4612-3730-3_11. PubMed DOI

Huang J., Ko P., Huang C., Wen P., Chen C., Shih M., Lin W., Huang F. Cytochrome P450 monooxygenase of Acanthamoeba castellanii participates in resistance to polyhexamethylene biguanide treatment. Parasite. 2021;28:77. doi: 10.1051/parasite/2021074. PubMed DOI PMC

She X., Zhang L., Peng J., Zhang J., Li H., Zhang P., Calderone R., Liu W., Li D. Mitochondrial Complex I Core Protein Regulates cAMP Signaling via Phosphodiesterase Pde2 and NAD Homeostasis in Candida albicans. Front. Microbiol. 2020;11:559975. doi: 10.3389/fmicb.2020.559975. PubMed DOI PMC

Taylor J., Yeomans A.M., Packham G. Targeted inhibition of mRNA translation initiation factors as a novel therapeutic strategy for mature B-cell neoplasms. Explor. Target. Anti-Tumor Ther. 2020;1:3–25. doi: 10.37349/etat.2020.00002. PubMed DOI PMC

Mo D., Liu C., Chen Y., Cheng X., Shen J., Zhao L., Zhang J. The mitochondrial ribosomal protein mRpL4 regulates Notch signaling. EMBO Rep. 2023;24:e55764. doi: 10.15252/embr.202255764. PubMed DOI PMC

Lindqvist L., Oberer M., Reibarkh M., Cencic R., Bordeleau M., Vogt E., Marintchev A., Tanaka J., Fagotto F., Altmann M., et al. Selective pharmacological targeting of a DEAD box RNA helicase. PLoS ONE. 2008;3:e1583. doi: 10.1371/journal.pone.0001583. PubMed DOI PMC

Zhang L., Li X. DEAD-Box RNA Helicases in Cell Cycle Control and Clinical Therapy. Cells. 2021;10:1540. doi: 10.3390/cells10061540. PubMed DOI PMC

Yildizhan H., Barkan N.P., Karahisar Turan S., Demiralp Ö., Özel Demiralp F.D., Uslu B., Ōzkan S.A. Chapter 1—Treatment strategies in cancer from past to present. In: Grumezescu A.M., editor. Drug Targeting and Stimuli Sensitive Drug Delivery Systems. William Andrew Publishing; Norwich, NY, USA: 2018. pp. 1–37.

Garcia-Diaz M., Bebenek K. Multiple functions of DNA polymerases. CRC Crit. Rev. Plant Sci. 2007;26:105–122. doi: 10.1080/07352680701252817. PubMed DOI PMC

Berdis A.J. Inhibiting DNA Polymerases as a Therapeutic Intervention against Cancer. Front. Mol. Biosci. 2017;4:78. doi: 10.3389/fmolb.2017.00078. PubMed DOI PMC

Martín-Navarro C.M., Lorenzo-Morales J., Cabrera-Serra M.G., Rancel F., Coronado-Álvarez N.M., Piñero J.E., Valladares B. The potential pathogenicity of chlorhexidine-sensitive Acanthamoeba strains isolated from contact lens cases from asymptomatic individuals in Tenerife, Canary Islands, Spain. J. Med. Microbiol. 2008;57:1399. doi: 10.1099/jmm.0.2008/003459-0. PubMed DOI

Sifaoui I., Reyes-Batlle M., López-Arencibia A., Wagner C., Chiboub O., De Agustino Rodríguez J., Rocha-Cabrera P., Valladares B., Piñero J.E., Lorenzo-Morales J. Evaluation of the anti-Acanthamoeba activity of two commercial eye drops commonly used to lower eye pressure. Exp. Parasitol. 2017;183:117–123. doi: 10.1016/j.exppara.2017.07.012. PubMed DOI

Sifaoui I., Reyes-Batlle M., López-Arencibia A., Chiboub O., Rodríguez-Martín J., Rocha-Cabrera P., Valladares B., Piñero J.E., Lorenzo-Morales J. Toxic effects of selected proprietary dry eye drops on Acanthamoeba. Sci. Rep. 2018;8:8520. doi: 10.1038/s41598-018-26914-3. PubMed DOI PMC

Rodríguez-Expósito R.L., Sifaoui I., Reyes-Batlle M., Fuchs F., Scheid P.L., Piñero J.E., Sutak R., Lorenzo-Morales J. Induction of Programmed Cell Death in Acanthamoeba culbertsoni by the Repurposed Compound Nitroxoline. Antioxidants. 2023;12:2081. doi: 10.3390/antiox12122081. PubMed DOI PMC

Arbon D., Zeniskova K., Subrtova K., Mach J., Stursa J., Machado M., Zahedifard F., Lestinova T., Hierro-Yap C., Neuzil J., et al. Repurposing of MitoTam: Novel Anti-Cancer Drug Candidate Exhibits Potent Activity against Major Protozoan and Fungal Pathogens. Antimicrob. Agents Chemother. 2022;66:e0072722. doi: 10.1128/aac.00727-22. PubMed DOI PMC

Hughes C.S., Moggridge S., Müller T., Sorensen P.H., Morin G.B., Krijgsveld J. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat. Protoc. 2019;14:68–85. doi: 10.1038/s41596-018-0082-x. PubMed DOI

Rappsilber J., Mann M., Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2007;2:1896–1906. doi: 10.1038/nprot.2007.261. PubMed DOI

Cox J., Hein M.Y., Luber C.A., Paron I., Nagaraj N., Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell Proteomics. 2014;13:2513–2526. doi: 10.1074/mcp.M113.031591. PubMed DOI PMC

Tyanova S., Temu T., Sinitcyn P., Carlson A., Hein M.Y., Geiger T., Mann M., Cox J. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods. 2016;13:731–740. doi: 10.1038/nmeth.3901. PubMed DOI