The simultaneous targeting of host and pathogen processes represents an untapped approach for the treatment of intracellular infections. Hypoxia-inducible factor-1 (HIF-1) is a host cell transcription factor that is activated by and required for the growth of the intracellular protozoan parasite Toxoplasma gondii at physiological oxygen levels. Parasite activation of HIF-1 is blocked by inhibiting the family of closely related Activin-Like Kinase (ALK) host cell receptors ALK4, ALK5, and ALK7, which was determined in part by use of an ALK4,5,7 inhibitor named SB505124. Besides inhibiting HIF-1 activation, SB505124 also potently blocks parasite replication under normoxic conditions. To determine whether SB505124 inhibition of parasite growth was exclusively due to inhibition of ALK4,5,7 or because the drug inhibited a second kinase, SB505124-resistant parasites were isolated by chemical mutagenesis. Whole-genome sequencing of these mutants revealed mutations in the Toxoplasma MAP kinase, TgMAPK1. Allelic replacement of mutant TgMAPK1 alleles into wild-type parasites was sufficient to confer SB505124 resistance. SB505124 independently impacts TgMAPK1 and ALK4,5,7 signaling since drug resistant parasites could not activate HIF-1 in the presence of SB505124 or grow in HIF-1 deficient cells. In addition, TgMAPK1 kinase activity is inhibited by SB505124. Finally, mice treated with SB505124 had significantly lower tissue burdens following Toxoplasma infection. These data therefore identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1.
References
[1]
Pereira-Chioccola VL, Vidal JE, Su C (2009) Toxoplasma gondii infection and cerebral toxoplasmosis in HIV-infected patients. Future Microbiol 4: 1363–1379.
[2]
Hill D, Dubey JP (2002) Toxoplasma gondii: transmission, diagnosis and prevention. Clin Microbiol Infect 8: 634–640.
[3]
Weiss LM, Kim K (2000) The development and biology of bradyzoites of Toxoplasma gondii. Front Biosci 5: D391–405.
[4]
Bohne W, Heesemann J, Gross U (1994) Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion. Infect Immun 62: 1761–1767.
[5]
Ferguson DJ, Hutchison WM (1987) The host-parasite relationship of Toxoplasma gondii in the brains of chronically infected mice. Virchows Arch A Pathol Anat Histopathol 411: 39–43.
[6]
Faucher B, Moreau J, Zaegel O, Franck J, Piarroux R (2011) Failure of conventional treatment with pyrimethamine and sulfadiazine for secondary prophylaxis of cerebral toxoplasmosis in a patient with AIDS. J Antimicrob Chemother 66: 1654–1656.
[7]
Dunay IR, Heimesaat MM, Bushrab FN, Muller RH, Stocker H, et al. (2004) Atovaquone maintenance therapy prevents reactivation of toxoplasmic encephalitis in a murine model of reactivated toxoplasmosis. Antimicrob Agents Chemother 48: 4848–4854.
[8]
Schaeffer M, Han SJ, Chtanova T, van Dooren GG, Herzmark P, et al. (2009) Dynamic imaging of T cell-parasite interactions in the brains of mice chronically infected with Toxoplasma gondii. J Immunol 182: 6379–6393.
[9]
Molestina RE, Payne TM, Coppens I, Sinai AP (2003) Activation of NF-{kappa}B by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated I{kappa}B to the parasitophorous vacuole membrane. J Cell Sci 116: 4359–4371.
[10]
Del Rio L, Butcher BA, Bennouna S, Hieny S, Sher A, et al. (2004) Toxoplasma gondii triggers myeloid differentiation factor 88-dependent IL-12 and chemokine ligand 2 (monocyte chemoattractant protein 1) responses using distinct parasite molecules and host receptors. J Immunol 172: 6954–6960.
[11]
Mason NJ, Fiore J, Kobayashi T, Masek KS, Choi Y, et al. (2004) TRAF6-dependent mitogen-activated protein kinase activation differentially regulates the production of interleukin-12 by macrophages in response to Toxoplasma gondii. Infect Immun 72: 5662–5667.
[12]
Yamamoto M, Ma JS, Mueller C, Kamiyama N, Saiga H, et al. (2011) ATF6{beta} is a host cellular target of the Toxoplasma gondii virulence factor ROP18. J Exp Med 208: 1533–1546.
[13]
Yamamoto M, Standley DM, Takashima S, Saiga H, Okuyama M, et al. (2009) A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3. J Exp Med 206: 2747–2760.
Saeij JP, Coller S, Boyle JP, Jerome ME, White MW, et al. (2007) Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue. Nature 445: 324–327.
[16]
Coppens I, Dunn JD, Romano JD, Pypaert M, Zhang H, et al. (2006) Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space. Cell 125: 261–274.
[17]
Romano JD, Sonda S, Bergbower E, Smith ME, Coppens I (2013) Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole. Molecular Biology of the Cell 24: 1974–1995.
[18]
Spear W, Chan D, Coppens I, Johnson RS, Giaccia A, et al. (2006) The host cell transcription factor hypoxia-inducible factor 1 is required for Toxoplasma gondii growth and survival at physiological oxygen levels. Cell Microbiol 8: 339–352.
[19]
Berra E, Benizri E, Ginouves A, Volmat V, Roux D, et al. (2003) HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia. EMBO J 22: 4082–4090.
[20]
Appelhoff RJ, Tian YM, Raval RR, Turley H, Harris AL, et al. (2004) Differential function of the prolyl hydroxylases PHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible factor. J Biol Chem 279: 38458–38465.
[21]
Wiley M, Sweeney KR, Chan DA, Brown KM, McMurtrey C, et al. (2010) Toxoplasma gondii activates hypoxia inducible factor by stabilizing the HIF-1 alpha subunit via type I activin like receptor kinase receptor signaling. J Biol Chem 285: 26976–26986.
[22]
DaCosta Byfield S, Major C, Laping NJ, Roberts AB (2004) SB-505124 is a selective inhibitor of transforming growth factor-beta type I receptors ALK4, ALK5, and ALK7. Mol Pharmacol 65: 744–752.
[23]
Wei S, Marches F, Daniel B, Sonda S, Heidenreich K, et al. (2002) Pyridinylimidazole p38 mitogen-activated protein kinase inhibitors block intracellular Toxoplasma gondii replication. Int J Parasitol 32: 969–977.
[24]
Brumlik MJ, Wei S, Finstad K, Nesbit J, Hyman LE, et al. (2004) Identification of a novel mitogen-activated protein kinase in Toxoplasma gondii. Int J Parasitol 34: 1245–1254.
[25]
Sugi T, Kobayashi K, Takemae H, Gong H, Ishiwa A, et al. (2013) Identification of mutations in TgMAPK1 of Toxoplasma gondii conferring resistance to 1NM-PP1. Int J Parasitol Drugs Drug Resist 3: 93–101.
[26]
Brumlik MJ, Pandeswara S, Ludwig SM, Jeansonne DP, Lacey MR, et al. (2013) TgMAPK1 is a Toxoplasma gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and interferon-gamma-mediated nitric oxide production. Exp Parasitol 134: 389–399.
[27]
Gum RJ, McLaughlin MM, Kumar S, Wang Z, Bower MJ, et al. (1998) Acquisition of sensitivity of stress-activated protein kinases to the p38 inhibitor, SB 203580, by alteration of one or more amino acids within the ATP binding pocket. J Biol Chem 273: 15605–15610.
[28]
Wilson KP, McCaffrey PG, Hsiao K, Pazhanisamy S, Galullo V, et al. (1997) The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAP kinase. Chem Biol 4: 423–431.
[29]
Tong L, Pav S, White DM, Rogers S, Crane KM, et al. (1997) A highly specific inhibitor of human p38 MAP kinase binds in the ATP pocket. Nat Struct Biol 4: 311–316.
[30]
Hunter CA, Bermudez L, Beernink H, Waegell W, Remington JS (1995) Transforming growth factor-beta inhibits interleukin-12-induced production of interferon-gamma by natural killer cells: a role for transforming growth factor-beta in the regulation of T cell-independent resistance to Toxoplasma gondii. Eur J Immunol 25: 994–1000.
[31]
Lourido S, Shuman J, Zhang C, Shokat KM, Hui R, et al. (2010) Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature 465: 359–362.
[32]
Sugi T, Kato K, Kobayashi K, Watanabe S, Kurokawa H, et al. (2010) Use of the kinase inhibitor analog 1NM-PP1 reveals a role for Toxoplasma gondii CDPK1 in the invasion step. Eukaryot Cell 9: 667–670.
[33]
Nathan C (2012) Fresh Approaches to Anti-Infective Therapies. Science Translational Medicine 4: 140sr142.
[34]
Ryan HE, Lo J, Johnson RS (1998) HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J 17: 3005–3015.
[35]
Radke JR, Guerini MN, Jerome M, White MW (2003) A change in the premitotic period of the cell cycle is associated with bradyzoite differentiation in Toxoplasma gondii. Mol Biochem Parasitol 131: 119–127.
[36]
Radke JR, Striepen B, Guerini MN, Jerome ME, Roos DS, et al. (2001) Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii. Mol Biochem Parasitol 115: 165–175.
[37]
MacDonald ML, Lamerdin J, Owens S, Keon BH, Bilter GK, et al. (2006) Identifying off-target effects and hidden phenotypes of drugs in human cells. Nat Chem Biol 2: 329–337.
[38]
Wermuth CG (2006) Selective optimization of side activities: the SOSA approach. Drug Discov Today 11: 160–164.
[39]
Imperi F, Massai F, Facchini M, Frangipani E, Visaggio D, et al. (2013) Repurposing the antimycotic drug flucytosine for suppression of Pseudomonas aeruginosa pathogenicity. Proceedings of the National Academy of Sciences 110: 7458–7463.
[40]
Debnath A, Parsonage D, Andrade RM, He C, Cobo ER, et al. (2012) A high-throughput drug screen for Entamoeba histolytica identifies a new lead and target. Nat Med 18: 956–960.
[41]
Patel G, Karver CE, Behera R, Guyett P, Sullenberger C, et al. (2013) Kinase scaffold repurposing for neglected disease drug discovery: Discovery of an efficacious, lapatanib-derived lead compound for trypanosomiasis. Journal of Medicinal Chemistry 56: 3820–3832.
[42]
Pfefferkorn ER, Pfefferkorn LC (1977) Toxoplasma gondii: characterization of a mutant resistant to 5- fluorodeoxyuridine. Exp Parasitol 42: 44–55.
[43]
Black MW, Arrizabalaga G, Boothroyd JC (2000) Ionophore-resistant mutants of Toxoplasma gondii reveal host cell permeabilization as an early event in egress. Mol Cell Biol 20: 9399–9408.
[44]
Smith DR, Quinlan AR, Peckham HE, Makowsky K, Tao W, et al. (2008) Rapid whole-genome mutational profiling using next-generation sequencing technologies. Genome Res 18: 1638–1642.
[45]
Hillier LW, Marth GT, Quinlan AR, Dooling D, Fewell G, et al. (2008) Whole-genome sequencing and variant discovery in C. elegans. Nat Methods 5: 183–188.
[46]
Garrison E, Marth GT (2012) Haplotype-based variant detection from short-read sequencing. ARXIV eprint arXiv: 1207.3907 Available: http://arxiv.org/abs/1207.3907. Accessed 30 April 2014.
[47]
Soete M, Camus D, Dubremetz JF (1994) Experimental induction of bradyzoite-specific antigen expression and cyst formation by the RH strain of Toxoplasma gondii in vitro. Exp Parasitol 78: 361–370.
[48]
Huynh MH, Carruthers VB (2009) Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot Cell 8: 530–539.
[49]
Soldati D, Boothroyd JC (1993) Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii. Science 260: 349–352.
[50]
Phelps E, Sweeney K, Blader IJ (2008) Toxoplasma gondii Rhoptry Discharge Correlates with Activation of the EGR2 Host Cell Transcription Factor. Infection and Immunity 76: 4703–4712.
