Babesiosis is a tick-transmitted zoonosis caused by apicomplexan parasites of the genus Babesia. Treatment of this emerging malaria-related disease has relied on antimalarial drugs and antibiotics. The proteasome of Plasmodium, the causative agent of malaria, has recently been validated as a target for anti-malarial drug development and therefore, in this study, we investigated the effect of epoxyketone (carfilzomib, ONX-0914 and epoxomicin) and boronic acid (bortezomib and ixazomib) proteasome inhibitors on the growth and survival of Babesia. Testing the compounds against Babesia divergens ex vivo revealed suppressive effects on parasite growth with activity that was higher than the cytotoxic effects on a non-transformed mouse macrophage cell line. Furthermore, we showed that the most-effective compound, carfilzomib, significantly reduces parasite multiplication in a Babesia microti infected mouse model without noticeable adverse effects. In addition, treatment with carfilzomib lead to an ex vivo and in vivo decrease in proteasome activity and accumulation of polyubiquitinated proteins compared to untreated control. Overall, our results demonstrate that the Babesia proteasome is a valid target for drug development and warrants the design of potent and selective B. divergens proteasome inhibitors for the treatment of babesiosis.

Department of Medicine University of California San Diego La Jolla USA

Institute of Parasitology Biology Centre of the Czech Academy of Sciences CZ 370 05 Ceske Budejovice Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences CZ 370 05 Ceske Budejovice Czech Republic; Faculty of Science University of South Bohemia CZ 370 05 Ceske Budejovice Czech Republic

Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego La Jolla USA

Zobrazit více v PubMed

Aboulaila M., Nakamura K., Govind Y., Yokoyama N., Igarashi I. Evaluation of the in vitro growth-inhibitory effect of epoxomicin on Babesia parasites. Vet. Parasitol. 2010;167:19–27. PubMed

Adams J. The proteasome: a suitable antineoplastic target. Nat. Rev. Canc. 2004;4:349–360. PubMed

Arastu-Kapur S., Anderl J.L., Kraus M., Parlati F., Shenk K.D., Lee S.J., Muchamuel T., Bennett M.K., Driessen C., Ball A.J., Kirk C.J. Nonproteasomal targets of the proteasome inhibitors bortezomib and carfilzomib: a link to clinical adverse events. Clin. Canc. Res. 2011;17:2734–2743. PubMed

Arisue N., Hashimoto T. Phylogeny and evolution of apicoplasts and apicomplexan parasites. Parasitol. Int. 2015;64:254–259. PubMed

Bedford L., Paine S., Sheppard P.W., Mayer R.J., Roelofs J. Assembly, structure, and function of the 26S proteasome. Trends Cell Biol. 2010;20:391–401. PubMed PMC

Benezra D., Brown A.E., Polsky B., Gold J.W., Armstrong D. Babesiosis and infection with human immunodeficiency virus (HIV) Ann. Intern. Med. 1987;107:944. PubMed

Bibo-Verdugo B., Jiang Z., Caffrey C.R., O'Donoghue A.J. Targeting proteasomes in infectious organisms to combat disease. FEBS J. 2017;284:1503–1517. PubMed

Bock R., Jackson L., de Vos A., Jorgensen W. Babesiosis of cattle. Parasitology. 2004;129(Suppl. l):S247–S269. PubMed

Burki F., Inagaki Y., Bråte J., Archibald J.M., Keeling P.J., Cavalier-Smith T., Sakaguchi M., Hashimoto T., Horak A., Kumar S., Klaveness D., Jakobsen K.S., Pawlowski J., Shalchian-Tabrizi K. Large-scale phylogenomic analyses reveal that two enigmatic protist lineages, telonemia and centroheliozoa, are related to photosynthetic chromalveolates. Genome Biol Evol. 2009;1:231–238. PubMed PMC

de Vries E., Corton C., Harris B., Cornelissen A.W., Berriman M. Expressed sequence tag (EST) analysis of the erythrocytic stages of Babesia bovis. Vet. Parasitol. 2006;138:61–74. PubMed

Demo S.D., Kirk C.J., Aujay M.A., Buchholz T.J., Dajee M., Ho M.N., Jiang J., Laidig G.J., Lewis E.R., Parlati F., Shenk K.D., Smyth M.S., Sun C.M., Vallone M.K., Woo T.M., Molineaux C.J., Bennett M.K. Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome. Canc. Res. 2007;67:6383–6391. PubMed

Dogovski C., Xie S.C., Burgio G., Bridgford J., Mok S., McCaw J.M., Chotivanich K., Kenny S., Gnädig N., Straimer J., Bozdech Z., Fidock D.A., Simpson J.A., Dondorp A.M., Foote S., Klonis N., Tilley L. Targeting the cell stress response of Plasmodium falciparum to overcome artemisinin resistance. PLoS Biol. 2015;13 PubMed PMC

Falagas M.E., Klempner M.S. Babesiosis in patients with AIDS: a chronic infection presenting as fever of unknown origin. Clin. Infect. Dis. 1996;22:809–812. PubMed

Ferrington D.A., Gregerson D.S. Immunoproteasomes: structure, function, and antigen presentation. Prog Mol Biol Transl Sci. 2012;109:75–112. PubMed PMC

Froberg M.K., Dannen D., Bakken J.S. Babesiosis and HIV. Lancet. 2004;363:704. PubMed

Giguere C.J., Schnellmann R.G. Limitations of SLLVY-AMC in calpain and proteasome measurements. Biochem. Biophys. Res. Commun. 2008;371:578–581. PubMed PMC

Gohil S., Herrmann S., Günther S., Cooke B.M. Bovine babesiosis in the 21st century: advances in biology and functional genomics. Int. J. Parasitol. 2013;43:125–132. PubMed

Groll M., Berkers C.R., Ploegh H.L., Ovaa H. Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome. Structure. 2006;14:451–456. PubMed

Gu Y., Bouwman P., Greco D., Saarela J., Yadav B., Jonkers J., Kuznetsov S.G. Suppression of BRCA1 sensitizes cells to proteasome inhibitors. Cell Death Dis. 2014;5 PubMed PMC

Haapasalo K., Suomalainen P., Sukura A., Siikamaki H., Jokiranta T.S. Fatal babesiosis in man, Finland, 2004. Emerg. Infect. Dis. 2010;16:1116–1118. PubMed PMC

Hildebrandt A., Gray J.S., Hunfeld K.P. Human babesiosis in Europe: what clinicians need to know. Infection. 2013;41:1057–1072. PubMed

Häselbarth K., Tenter A.M., Brade V., Krieger G., Hunfeld K.P. First case of human babesiosis in Germany - clinical presentation and molecular characterisation of the pathogen. Int J Med Microbiol. 2007;297:197–204. PubMed

Janouskovec J., Horák A., Oborník M., Lukes J., Keeling P.J. A common red algal origin of the apicomplexan, dinoflagellate, and heterokont plastids. Proc. Natl. Acad. Sci. U. S. A. 2010;107:10949–10954. PubMed PMC

Kane R.C., Bross P.F., Farrell A.T., Pazdur R. Velcade: U.S. FDA approval for the treatment of multiple myeloma progressing on prior therapy. Oncology. 2003;8:508–513. PubMed

Kane R.C., Farrell A.T., Sridhara R., Pazdur R. United States Food and Drug Administration approval summary: bortezomib for the treatment of progressive multiple myeloma after one prior therapy. Clin. Canc. Res. 2006;12:2955–2960. PubMed

Khare S., Nagle A.S., Biggart A., Lai Y.H., Liang F., Davis L.C., Barnes S.W., Mathison C.J., Myburgh E., Gao M.Y., Gillespie J.R., Liu X., Tan J.L., Stinson M., Rivera I.C., Ballard J., Yeh V., Groessl T., Federe G., Koh H.X., Venable J.D., Bursulaya B., Shapiro M., Mishra P.K., Spraggon G., Brock A., Mottram J.C., Buckner F.S., Rao S.P., Wen B.G., Walker J.R., Tuntland T., Molteni V., Glynne R.J., Supek F. Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness. Nature. 2016;537:229–233. PubMed PMC

Kim K.B., Crews C.M. From epoxomicin to carfilzomib: chemistry, biology, and medical outcomes. Nat. Prod. Rep. 2013;30:600–604. PubMed PMC

Kish-Trier E., Hill C.P. Structural biology of the proteasome. Annu. Rev. Biophys. 2013;42:29–49. PubMed PMC

Kisselev A.F., van der Linden W.A., Overkleeft H.S. Proteasome inhibitors: an expanding army attacking a unique target. Chem. Biol. 2012;19:99–115. PubMed PMC

Krause P.J., Gewurz B.E., Hill D., Marty F.M., Vannier E., Foppa I.M., Furman R.R., Neuhaus E., Skowron G., Gupta S., McCalla C., Pesanti E.L., Young M., Heiman D., Hsue G., Gelfand J.A., Wormser G.P., Dickason J., Bia F.J., Hartman B., Telford S.R., Christianson D., Dardick K., Coleman M., Girotto J.E., Spielman A. Persistent and relapsing babesiosis in immunocompromised patients. Clin. Infect. Dis. 2008;46:370–376. PubMed

Kuhn D.J., Chen Q., Voorhees P.M., Strader J.S., Shenk K.D., Sun C.M., Demo S.D., Bennett M.K., van Leeuwen F.W., Chanan-Khan A.A., Orlowski R.Z. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma. Blood. 2007;110:3281–3290. PubMed PMC

Kupperman E., Lee E.C., Cao Y., Bannerman B., Fitzgerald M., Berger A., Yu J., Yang Y., Hales P., Bruzzese F., Liu J., Blank J., Garcia K., Tsu C., Dick L., Fleming P., Yu L., Manfredi M., Rolfe M., Bolen J. Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer. Canc. Res. 2010;70:1970–1980. PubMed

LaMonte G.M., Almaliti J., Bibo-Verdugo B., Keller L., Zou B.Y., Yang J., Antonova-Koch Y., Orjuela-Sanchez P., Boyle C.A., Vigil E., Wang L., Goldgof G.M., Gerwick L., O'Donoghue A.J., Winzeler E.A., Gerwick W.H., Ottilie S. Development of a potent inhibitor of the Plasmodium proteasome with reduced mammalian toxicity. J. Med. Chem. 2017;60:6721–6732. PubMed PMC

Lantos P.M., Krause P.J. Babesiosis: similar to malaria but different. Pediatr. Ann. 2002;31:192–197. PubMed

Leiby D.A. Transfusion-transmitted Babesia spp.: bull's-eye on Babesia microti. Clin. Microbiol. Rev. 2011;24:14–28. PubMed PMC

Lemieux J.E., Tran A.D., Freimark L., Schaffner S.F., Goethert H., Andersen K.G., Bazner S., Li A., McGrath G., Sloan L., Vannier E., Milner D., Pritt B., Rosenberg E., Telford S., Bailey J.A., Sabeti P.C. A global map of genetic diversity in Babesia microti reveals strong population structure and identifies variants associated with clinical relapse. Nat Microbiol. 2016;1:16079. PubMed PMC

Li H., Bogyo M., da Fonseca P.C. The cryo-EM structure of the Plasmodium falciparum 20S proteasome and its use in the fight against malaria. FEBS J. 2016;283:4238–4243. PubMed PMC

Li H., O'Donoghue A.J., van der Linden W.A., Xie S.C., Yoo E., Foe I.T., Tilley L., Craik C.S., da Fonseca P.C., Bogyo M. Structure- and function-based design of Plasmodium-selective proteasome inhibitors. Nature. 2016;530:233–236. PubMed PMC

Li H., Ponder E.L., Verdoes M., Asbjornsdottir K.H., Deu E., Edgington L.E., Lee J.T., Kirk C.J., Demo S.D., Williamson K.C., Bogyo M. Validation of the proteasome as a therapeutic target in Plasmodium using an epoxyketone inhibitor with parasite-specific toxicity. Chem. Biol. 2012;19:1535–1545. PubMed PMC

Lobo C.A., Cursino-Santos J.R., Alhassan A., Rodrigues M. Babesia: an emerging infectious threat in transfusion medicine. PLoS Pathog. 2013;9 PubMed PMC

Malandrin L., L'Hostis M., Chauvin A. Isolation of Babesia divergens from Carrier cattle blood using in vitro culture. Vet. Res. 2004;35:131–139. PubMed

Muchamuel T., Basler M., Aujay M.A., Suzuki E., Kalim K.W., Lauer C., Sylvain C., Ring E.R., Shields J., Jiang J., Shwonek P., Parlati F., Demo S.D., Bennett M.K., Kirk C.J., Groettrup M. A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nat. Med. 2009;15:781–787. PubMed

Mørch K., Holmaas G., Frolander P.S., Kristoffersen E.K. Severe human Babesia divergens infection in Norway. Int. J. Infect. Dis. 2015;33:37–38. PubMed

Ord R.L., Lobo C.A. Human babesiosis: pathogens, prevalence, diagnosis and treatment. Curr. Clin. Microbiol. Rep. 2015;2:173–181. PubMed PMC

Perner J., Sobotka R., Sima R., Konvickova J., Sojka D., Oliveira P.L., Hajdusek O., Kopacek P. Acquisition of exogenous haem is essential for tick reproduction. Elife. 2016;5 PubMed PMC

Rosner F., Zarrabi M.H., Benach J.L., Habicht G.S. Babesiosis in splenectomized adults. Review of 22 reported cases. Am. J. Med. 1984;76:696–701. PubMed

Schnittger L., Rodriguez A.E., Florin-Christensen M., Morrison D.A. Babesia: a world emerging. Infect. Genet. Evol. 2012;12:1788–1809. PubMed

Schreeg M.E., Marr H.S., Tarigo J.L., Cohn L.A., Bird D.M., Scholl E.H., Levy M.G., Wiegmann B.M., Birkenheuer A.J. Mitochondrial genome sequences and structures aid in the resolution of piroplasmida phylogeny. PLoS One. 2016;11 PubMed PMC

Simon M.S., Westblade L.F., Dziedziech A., Visone J.E., Furman R.R., Jenkins S.G., Schuetz A.N., Kirkman L.A. Clinical and molecular evidence of atovaquone and azithromycin resistance in relapsed Babesia microti infection associated with rituximab and chronic lymphocytic leukemia. Clin. Infect. Dis. 2017;65:1222–1225. PubMed PMC

Stowell C.P., Gelfand J.A., Shepard J.A., Kratz A. Case records of the Massachusetts General Hospital. Case 17-2007. A 25-year-old woman with relapsing fevers and recent onset of dyspnea. N. Engl. J. Med. 2007;356:2313–2319. PubMed

Tomko R.J., Hochstrasser M. Molecular architecture and assembly of the eukaryotic proteasome. Annu. Rev. Biochem. 2013;82:415–445. PubMed PMC

Uhnoo I., Cars O., Christensson D., Nyström-Rosander C. First documented case of human babesiosis in Sweden. Scand. J. Infect. Dis. 1992;24:541–547. PubMed

Vannier E., Krause P.J. Human babesiosis. N. Engl. J. Med. 2012;366:2397–2407. PubMed

Vannier E.G., Diuk-Wasser M.A., Ben Mamoun C., Krause P.J. Babesiosis. Infect. Dis. Clin. 2015;29:357–370. PubMed PMC

Verdoes M., Florea B.I., Menendez-Benito V., Maynard C.J., Witte M.D., van der Linden W.A., van den Nieuwendijk A.M., Hofmann T., Berkers C.R., van Leeuwen F.W., Groothuis T.A., Leeuwenburgh M.A., Ovaa H., Neefjes J.J., Filippov D.V., van der Marel G.A., Dantuma N.P., Overkleeft H.S. A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo. Chem. Biol. 2006;13:1217–1226. PubMed

Vial H.J., Gorenflot A. Chemotherapy against babesiosis. Vet. Parasitol. 2006;138:147–160. PubMed

Voges D., Zwickl P., Baumeister W. The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 1999;68:1015–1068. PubMed

Wormser G.P., Prasad A., Neuhaus E., Joshi S., Nowakowski J., Nelson J., Mittleman A., Aguero-Rosenfeld M., Topal J., Krause P.J. Emergence of resistance to azithromycin-atovaquone in immunocompromised patients with Babesia microti infection. Clin. Infect. Dis. 2010;50:381–386. PubMed

Yabsley M.J., Shock B.C. Natural history of zoonotic Babesia: role of wildlife reservoirs. Int J Parasitol Parasites Wildl. 2013;2:18–31. PubMed PMC

Yang J., Wang Z., Fang Y., Jiang J., Zhao F., Wong H., Bennett M.K., Molineaux C.J., Kirk C.J. Pharmacokinetics, pharmacodynamics, metabolism, distribution, and excretion of carfilzomib in rats. Drug Metab. Dispos. 2011;39:1873–1882. PubMed

Zintl A., Mulcahy G., Skerrett H.E., Taylor S.M., Gray J.S. Babesia divergens, a bovine blood parasite of veterinary and zoonotic importance. Clin. Microbiol. Rev. 2003;16:622–636. PubMed PMC

Chelation of Mitochondrial Iron as an Antiparasitic Strategy

Establishment of a stable transfection and gene targeting system in Babesia divergens

Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors