Background Target repurposing utilizes knowledge of “druggable” targets obtained in one organism and exploits this information to pursue new potential drug targets in other organisms. Here we describe such studies to evaluate whether inhibitors targeting the kinase domain of the mammalian Target of Rapamycin (mTOR) and human phosphoinositide-3-kinases (PI3Ks) show promise against the kinetoplastid parasites Trypanosoma brucei, T. cruzi, Leishmania major, and L. donovani. The genomes of trypanosomatids encode at least 12 proteins belonging to the PI3K protein superfamily, some of which are unique to parasites. Moreover, the shared PI3Ks differ greatly in sequence from those of the human host, thereby providing opportunities for selective inhibition. Methodology/Principal Findings We focused on 8 inhibitors targeting mTOR and/or PI3Ks selected from various stages of pre-clinical and clinical development, and tested them against in vitro parasite cultures and in vivo models of infection. Several inhibitors showed micromolar or better efficacy against these organisms in culture. One compound, NVP-BEZ235, displayed sub-nanomolar potency, efficacy against cultured parasites, and an ability to clear parasitemia in an animal model of T. brucei rhodesiense infection. Conclusions/Significance These studies strongly suggest that mammalian PI3/TOR kinase inhibitors are a productive starting point for anti-trypanosomal drug discovery. Our data suggest that NVP-BEZ235, an advanced clinical candidate against solid tumors, merits further investigation as an agent for treating African sleeping sickness.
References
[1]
(2001) Human African trypanosomiasis: a guide for drug supply. World Health Organization.
[2]
Chappuis F, Sundar S, Hailu A, Ghalib H, Rijal S, et al. (2007) Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nat Rev Microbiol 5: 873–882. doi: 10.1038/nrmicro1748
[3]
Castro JA, de Mecca MM, Bartel LC (2006) Toxic side effects of drugs used to treat Chagas' disease (American trypanosomiasis). Hum Exp Toxicol 25: 471–479. doi: 10.1191/0960327106het653oa
[4]
de Koning HP (2008) Ever-increasing complexities of diamidine and arsenical crossresistance in African trypanosomes. Trends Parasitol 24: 345–349. doi: 10.1016/j.pt.2008.04.006
[5]
Secor WE, Nguyen-Dinh P (2007) Mechanisms of resistance to antiparasitic agents. Man Clin Microbiol (9th Ed) 2: 2240–2249.
[6]
Wilkinson SR, Taylor MC, Horn D, Kelly JM, Cheeseman I (2008) A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes. Proc Natl Acad Sci U S A, Early Ed 1–6.6
[7]
Nwaka S, Hudson A (2006) Innovative lead discovery strategies for tropical diseases. Nat Rev Drug Discov 5: 941–955. doi: 10.1038/nrd2144
[8]
Frearson JA, Brand S, McElroy SP, Cleghorn LAT, Smid O, et al. (2010) N-myristoyltransferase inhibitors as new leads to treat sleeping sickness. Nature 464: 728–732. doi: 10.1038/nature08893
[9]
Andrews KT, Walduck A, Kelso MJ, Fairlie DP, Saul A, et al. (2000) Anti-malarial effect of histone deacetylation inhibitors and mammalian tumour cytodifferentiating agents. International Journal for Parasitology 30: 761–768. doi: 10.1016/S0020-7519(00)00043-6
[10]
Eastman RT, White J, Hucke O, Bauer K, Yokoyama K, et al. (2005) Resistance to a Protein Farnesyltransferase Inhibitor in Plasmodium falciparum. Journal of Biological Chemistry 280: 13554–13559. doi: 10.1074/jbc.M413556200
[11]
Campbell RK, Pollastri MP (2011) Target Repurposing for Neglected Diseases. Future Medicinal Chemistry. in press. doi: 10.4155/fmc.11.92
[12]
Hopkins AL, Groom CR (2002) The druggable genome. Nat Rev Drug Discov 1: 727–730. doi: 10.1038/nrd892
[13]
Ihle NT, Powis G (2009) Take your PIK: phosphatidylinositol 3-kinase inhibitors race through the clinic and toward cancer therapy. Mol Cancer Ther 8: 1–9. doi: 10.1158/1535-7163.MCT-08-0801
[14]
Harris SJ, Foster JG, Ward SG (2009) PI3K isoforms as drug targets in inflammatory diseases: lessons from pharmacological and genetic strategies. Curr Opin Invest Drugs (BioMed Cent) 10: 1151–1162.
Choi J, Chen J, Schreiber SL, Clardy J (1996) Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science 273: 239–242. doi: 10.1126/science.273.5272.239
[17]
Liang J, Choi J, Clardy J (1999) Refined structure of the FKBP12-rapamycin-FRB ternary complex at 2.2 A resolution. Acta Crystallogr D Biol Crystallogr 55: 736–744. doi: 10.1107/S0907444998014747
[18]
Albert S, Serova M, Dreyer C, Sablin M-P, Faivre S, et al. (2010) New inhibitors of the mammalian target of rapamycin signaling pathway for cancer. Expert Opin Invest Drugs 19: 919–930. doi: 10.1517/13543784.2010.499121
[19]
Feldman ME, Apsel B, Uotila A, Loewith R, Knight ZA, et al. (2009) Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol 7: e38. doi: 10.1371/journal.pbio.1000038
[20]
Apsel B, Blair JA, Gonzalez B, Nazif TM, Feldman ME, et al. (2008) Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases. Nat Chem Biol 4: 691–699. doi: 10.1038/nchembio.117
[21]
Zask A, Verheijen JC, Curran K, Kaplan J, Richard DJ, et al. (2009) ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin: Design and Synthesis of Highly Potent and Selective Pyrazolopyrimidines. J Med Chem 52: 5013–5016. doi: 10.1021/jm900851f
[22]
Garcia-Martinez JM, Moran J, Clarke RG, Gray A, Cosulich SC, et al. (2009) Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). Biochem J 421: 29–42. doi: 10.1042/BJ20090489
[23]
Malagu K, Duggan H, Menear K, Hummersone M, Gomez S, et al. (2009) The discovery and optimisation of pyrido[2,3-d]pyrimidine-2,4-diamines as potent and selective inhibitors of mTOR kinase. Bioorg Med Chem Lett 19: 5950–5953. doi: 10.1016/j.bmcl.2009.08.038
[24]
Nuss JM, Tsuhako AL, Anand NK (2009) Emerging therapies based on inhibitors of phosphatidyl-inositol-3-kinases. Annu Rep Med Chem 44: 339–356, 332 plates.
[25]
Guertin DA, Sabatini DM (2009) The pharmacology of mTOR inhibition. Sci Signal 2: pe24. doi: 10.1126/scisignal.267pe24
[26]
Verheijen J, Yu K, Zask A (2008) mTOR Inhibitors in Oncology. Annu Rep Med Chem 43: 189–202.
[27]
Bahia D, Oliveira LM, Lima FM, Oliveira P, Silveira JF, et al. (2009) The TryPIKinome of five human pathogenic trypanosomatids: Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Leishmania braziliensis and Leishmania infantum–new tools for designing specific inhibitors. Biochem Biophys Res Commun 390: 963–970. doi: 10.1016/j.bbrc.2009.10.086
[28]
Brown JR, Auger KRPhylogenomics of phosphoinositide lipid kinases: perspectives on the evolution of second messenger signaling and drug discovery. BMC Evol Biol 11: 4. doi: 10.1186/1471-2148-11-4
[29]
Hall BS, Gabernet-Castello C, Voak A, Goulding D, Natesan SK, et al. (2006) TbVps34, the trypanosome orthologue of Vps34, is required for Golgi complex segregation. J Biol Chem 281: 27600–27612. doi: 10.1074/jbc.M602183200
[30]
Barquilla A, Crespo JL, Navarro M (2008) Rapamycin inhibits trypanosome cell growth by preventing TOR complex 2 formation. Proc Natl Acad Sci U S A 105: 14579–14584. doi: 10.1073/pnas.0802668105
[31]
Madeira da Silva L, Beverley SM (2010) Expansion of the target of rapamycin (TOR) kinase family and function in Leishmania shows that TOR3 is required for acidocalcisome biogenesis and animal infectivity. Proc Natl Acad Sci U S A 107: 11965–11970. doi: 10.1073/pnas.1004599107
[32]
de Jesus TC, Tonelli RR, Nardelli SC, da Silva Augusto L, Motta MC, et al. (2010) Target of rapamycin (TOR)-like 1 kinase is involved in the control of polyphosphate levels and acidocalcisome maintenance in Trypanosoma brucei. J Biol Chem 285: 24131–24140. doi: 10.1074/jbc.M110.120212
[33]
Polak P, Hall MN (2009) mTOR and the control of whole body metabolism. Curr Opin Cell Biol 21: 209–218. doi: 10.1016/j.ceb.2009.01.024
[34]
Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo JL, et al. (2002) Two TOR Complexes, Only One of which Is Rapamycin Sensitive, Have Distinct Roles in Cell Growth Control. Molecular cell 10: 457–468. doi: 10.1016/S1097-2765(02)00636-6
[35]
Soulard A, Cohen A, Hall MN (2009) TOR signaling in invertebrates. Curr Opin Cell Biol 21: 825–836. doi: 10.1016/j.ceb.2009.08.007
[36]
Barquilla A, Navarro M (2009) Trypanosome TOR complex 2 functions in cytokinesis. Cell Cycle 8: 697–699. doi: 10.4161/cc.8.5.7808
[37]
Madeira da Silva L, Owens KL, Murta SM, Beverley SM (2009) Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins. Proc Natl Acad Sci U S A 106: 7583–7588. doi: 10.1073/pnas.0901698106
[38]
Turner CM, McLellan S, Lindergard LA, Bisoni L, Tait A, et al. (2004) Human infectivity trait in Trypanosoma brucei: stability, heritability and relationship to sra expression. Parasitology 129: 445–454. doi: 10.1017/S0031182004005906
[39]
Onyango JD, Burri C, Brun R (2000) An automated biological assay to determine levels of the trypanocidal drug melarsoprol in biological fluids. Acta Trop 74: 95–100. doi: 10.1016/S0001-706X(99)00080-7
[40]
Raz B, Iten M, Grether-Buhler Y, Kaminsky R, Brun R (1997) The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro. Acta Trop 68: 139–147. doi: 10.1016/S0001-706X(97)00079-X
[41]
Buckner FS, Verlinde CL, La Flamme AC, Van Voorhis WC (1996) Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase. Antimicrob Agents Chemother 40: 2592–2597.
[42]
Kapler GM, Coburn CM, Beverley SM (1990) Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol Cell Biol 10: 1084–1094.
[43]
Goyard S, Segawa H, Gordon J, Showalter M, Duncan R, et al. (2003) An in vitro system for developmental and genetic studies of Leishmania donovani phosphoglycans. Mol Biochem Parasitol 130: 31–42. doi: 10.1016/S0166-6851(03)00142-7
[44]
Akopyants NS, Kimblin N, Secundino N, Patrick R, Peters N, et al. (2009) Demonstration of genetic exchange during cyclical development of Leishmania in the sand fly vector. Science 324: 265–268. doi: 10.1126/science.1169464
[45]
Cruz AK, Titus R, Beverley SM (1993) Plasticity in chromosome number and testing of essential genes in Leishmania by targeting. Proc Natl Acad Sci U S A 90: 1599–1603. doi: 10.1073/pnas.90.4.1599
[46]
Maira SM, Stauffer F, Brueggen J, Furet P, Schnell C, et al. (2008) Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity. Mol Cancer Ther 7: 1851–1863. doi: 10.1158/1535-7163.MCT-08-0017
[47]
Thalhofer CJ, Graff JW, Love-Homan L, Hickerson SM, Craft N, et al. (2010) In vivo imaging of transgenic Leishmania parasites in a live host. J Vis Exp.
[48]
Spath GF, Beverley SM (2001) A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation. Exp Parasitol 99: 97–103. doi: 10.1006/expr.2001.4656
[49]
Yu K, Toral-Barza L, Shi C, Zhang WG, Lucas J, et al. (2009) Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin. Cancer Res 69: 6232–6240. doi: 10.1158/0008-5472.CAN-09-0299
[50]
Garlich JR, De P, Dey N, Su JD, Peng X, et al. (2008) A vascular targeted pan phosphoinositide 3-kinase inhibitor prodrug, SF1126, with antitumor and antiangiogenic activity. Cancer Res 68: 206–215. doi: 10.1158/0008-5472.CAN-07-0669
[51]
Kristof AS, Pacheco-Rodriguez G, Schremmer B, Moss J (2005) LY303511 (2-piperazinyl-8-phenyl-4H-1-benzopyran-？4-one)acts via phosphatidylinositol 3-kinase-independent pathways to inhibit cell proliferation via mammalian target of rapamycin (mTOR)- and non-mTOR-dependent mechanisms. J Pharmacol Exp Ther 314: 1134–1143. doi: 10.1124/jpet.105.083550
[52]
Ding J, Vlahos C, Liu R, Brown R, Badwey J (1995) Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils. J Biol Chem 270: 11684–11691. doi: 10.1074/jbc.270.19.11684
[53]
Fan QW, Cheng CK, Nicolaides TP, Hackett CS, Knight ZA, et al. (2007) A dual phosphoinositide-3-kinase alpha/mTOR inhibitor cooperates with blockade of epidermal growth factor receptor in PTEN-mutant glioma. Cancer Res 67: 7960–7965. doi: 10.1158/0008-5472.CAN-07-2154
[54]
Fan QW, Knight ZA, Goldenberg DD, Yu W, Mostov KE, et al. (2006) A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma. Cancer Cell 9: 341–349. doi: 10.1016/j.ccr.2006.03.029
[55]
Knight ZA, Gonzalez B, Feldman ME, Zunder ER, Goldenberg DD, et al. (2006) A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling. Cell 125: 733–747. doi: 10.1016/j.cell.2006.03.035
[56]
Ballou LM, Selinger ES, Choi JY, Drueckhammer DG, Lin RZ (2007) Inhibition of mammalian target of rapamycin signaling by 2-(morpholin-1-yl)pyrimido[2,1-alpha]iso？quinolin-4-one. J Biol Chem 282: 24463–24470. doi: 10.1074/jbc.M704741200
[57]
McMillin DW, Ooi M, Delmore J, Negri J, Hayden P, et al. (2009) Antimyeloma activity of the orally bioavailable dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235. Cancer Res 69: 5835–5842. doi: 10.1158/0008-5472.CAN-08-4285
[58]
Liu TJ, Koul D, LaFortune T, Tiao N, Shen RJ, et al. (2009) NVP-BEZ235, a novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, elicits multifaceted antitumor activities in human gliomas. Mol Cancer Ther 8: 2204–2210. doi: 10.1158/1535-7163.MCT-09-0160
[59]
Tsang CK, Qi H, Liu LF, Zheng XF (2007) Targeting mammalian target of rapamycin (mTOR) for health and diseases. Drug Discov Today 12: 112–124. doi: 10.1016/j.drudis.2006.12.008
[60]
Robinson DR, Sherwin T, Ploubidou A, Byard EH, Gull K (1995) Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle. J Cell Biol 128: 1163–1172. doi: 10.1083/jcb.128.6.1163
[61]
Wenzler T, Boykin DW, Ismail MA, Hall JE, Tidwell RR, et al. (2009) New treatment option for second-stage African sleeping sickness: in vitro and in vivo efficacy of aza analogs of DB289. Antimicrob Agents Chemother 53: 4185–4192. doi: 10.1128/AAC.00225-09
[62]
Cheekatla S, Aggarwal A, Naik S (2011) mTOR signaling pathway regulates the IL-12/IL-10 axis in Leishmania donovani infection. Medical Microbiology and Immunology 1–10. doi: 10.1007/s00430-010-0177-7
[63]
Seifert K, Munday J, Syeda T, Croft SL (2011) In vitro interactions between sitamaquine and amphotericin B, sodium stibogluconate, miltefosine, paromomycin and pentamidine against Leishmania donovani. J Antimicrob Chemother 66: 850–854. doi: 10.1093/jac/dkq542
[64]
Kim D-H, Sarbassov DD, Ali SM, King JE, Latek RR, et al. (2002) mTOR Interacts with Raptor to Form a Nutrient-Sensitive Complex that Signals to the Cell Growth Machinery. Cell 110: 163–175. doi: 10.1016/S0092-8674(02)00808-5
[65]
Hara K, Maruki Y, Long X, Yoshino K-i, Oshiro N, et al. (2002) Raptor, a Binding Partner of Target of Rapamycin (TOR), Mediates TOR Action. Cell 110: 177–189. doi: 10.1016/S0092-8674(02)00833-4
[66]
Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, et al. (2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 14: 1296–1302. doi: 10.1016/j.cub.2004.06.054
[67]
Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, et al. (2004) Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 6: 1122–1128. doi: 10.1038/ncb1183
[68]
Hayakawa M, Kaizawa H, Moritomo H, Koizumi T, Ohishi T, et al. (2007) Synthesis and biological evaluation of pyrido[3′,2′:4,5]furo[3,2-d]pyrimidine derivatives as novel PI3 kinase p110alpha inhibitors. Bioorg Med Chem Lett 17: 2438–2442. doi: 10.1016/j.bmcl.2007.02.032
[69]
Brunn GJ, Williams J, Sabers C, Wiederrecht G, Lawrence JC Jr, et al. (1996) Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J 15: 5256–5267.
[70]
Vlahos CJ, Matter WF, Hui KY, Brown RF (1994) A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyr？an-4-one(LY294002). J Biol Chem 269: 5241–5248.
[71]
Griffin RJ, Fontana G, Golding BT, Guiard S, Hardcastle IR, et al. (2005) Selective benzopyranone and pyrimido[2,1-a]isoquinolin-4-one inhibitors of DNA-dependent protein kinase: synthesis, structure-activity studies, and radiosensitization of a human tumor cell line in vitro. J Med Chem 48: 569–585. doi: 10.1021/jm049526a
