The cell cycle as a therapeutic target against Trypanosoma brucei: Hesperadin inhibits Aurora kinase-1 and blocks mitotic progression in bloodstream forms

Abstract

Aurora kinase family members co-ordinate a range of events associated with mitosis and cytokinesis. Anti-cancer therapies are currently being developed against them. Here, we evaluate whether Aurora kinase-1 (TbAUK1) from pathogenic Trypanosoma brucei might be targeted in anti-parasitic therapies as well. Conditional knockdown of TbAUK1 within infected mice demonstrated its essential contribution to infection. An in vitro kinase assay was developed which used recombinant trypanosome histone H3 as a substrate. Tandem mass spectroscopy identified a novel phosphorylation site in the carboxyl-tail of recombinant trypanosome histone H3. Hesperadin, an inhibitor of human Aurora B, prevented the phosphorylation of substrate with IC(50) of 40 nM. Growth of cultured bloodstream forms was also sensitive to Hesperadin (IC(50) of 50 nM). Hesperadin blocked nuclear division and cytokinesis but not other aspects of the cell cycle. Consequently, growth arrested cells accumulated multiple kinetoplasts, flagella and nucleoli, similar to the effects of RNAi-dependent knockdown of TbAUK1 in cultured bloodstream forms cells. Molecular models predicted high-affinity binding of Hesperadin to both conserved and novel sites in TbAUK1. Collectively, these data demonstrate that cell cycle progression is essential for infections with T. brucei and that parasite Aurora kinases can be targeted with small-molecule inhibitors.

Fig. 1

TbAU1K is essential for infection in mice. Mice were inoculated ip with 3×10⁶ TbAUK1 RNAi cells. One group containing three mice received 1 mg/ml doxycycline in the water at day 0 (+ Dox). The control group comprised of two mice received water without doxycycline (- Dox). (A) Parasiemia was monitored in peripheral blood at the times indicated. Each line plots the infection in a single mouse. The crosses indicate that the mice died within 24 hours of the last recorded parasitemia. The detection limit of the assay is 2×10⁵ cells/ml. (B) After 3 days of growth in a separate doxycycline treated mouse, trypanosomes were examined in the blood by DIC, or were labeled with antibodies against PFR (red) and counterstained with DAPI (blue). The cells at upper right were viewed at 60× while cells in the lower panels were viewed at 100×. The lower right panel shows two cells, each of which has multiple flagella and multiple kinetoplasts. The bars are size markers of 10 μm. Substrate specificities of TbAUK1 and TbAUK3.

Fig. 2

In vitro kinase assay of TbAUK1. (A) Constitutive expression of AU1-tagged TbAUK1. Parental AnTat1.1 PF were transformed with the constitutive expression vector pTSA-AU1.TbAUK. When pulled down with anti-AU1 Sepharose beads, transformants phosphorylated myelin basic protein (MBP), in a manner that was inhibited by Hesperadin. Parental cells phosphorylated MBP at a background level. (B) Inducible expression of AU1-tagged wild-type TbAUK1 and the K58R mutated TbAUK1. The tagged proteins were detected in cell homogenates by western blot with antibodies against AU1 (upper panels). TbRACK1 was used as a loading control (middle panels). The immunoprecipitated proteins phosphorylated MBP (lower panel). (C) Nucleotide specificity of TbAUK1. Unlabeled nucleotides (1 mM each) were added to the standard reaction mix. Only unlabeled ATP prevented phosphorylation of MBP.

Fig. 3

TbAUK1 phosphorylates trypanosome histones. (A) TbAUK1 can phosphorylate mammalian histone H3 and MBP, but not H1. Coomassie stain reveals equivalent substrate in each reaction. (B) Phosphorylation of endogenous histones by TbAUK1. Trypanosome histones were acid extracted from a particulate fraction and TbH3, TbH2A and TbH2B were tentatively identified based upon their molecular weights of 14.7 kDa, 14.2 kDa and 12.5 kDa, respectively. The histones were incubated with immunoprecipitated proteins from control cultures (AnTat); or from cultures expressing AU1.TbAUK1. TbAUK1 phosphorylated proteins with sizes equivalent to H3 and H2B. (C) TbAUK1 can phosphorylate recombinant TbH3 and TbH2B. TbH3 (Tb927.1.2530) and TbH2B (Tb10.406.0350) were amplified from genomic DNA, cloned in pQE80 and used as substrate in assays where the kinase source was either precipitated from control cultures (AnTat), or precipitated from cultures expressing AU1.TbAU1K.

Fig. 4

Identification of phosphorylation sites within TbH3 and TbH2B. (A) Sequence alignments of histone H3 and H2B from human (Hs), yeast (Sc) and T. brucei (Tb). Phosphorylation sites are blocked in black. (B) LC/MS/MS spectra of the triply-charged phosphorylated peptides detected in TbH3 and TbH2B. TbH3 digested with AspN endopeptidase. Peptide DTNRACIHSGRVT(p)IQPK (amino acids 104–120) had a mass-to-charge ratio (m/z) of 678.3. The MS/MS spectrum revealed multiple fragment ions including neutral loss fragments [M-H₃PO₄+3H]³⁺ at m/z 645.6. Wild-type b11²⁺ was detected, together with [b13-18]²⁺, [b14-18]²⁺, [b15-18]²⁺ and [b13+18]²⁺. The b-ion series indicated that the phosphorylation occurred at Thr116. TbH2B was digested with ArgC endopeptidase. Triply charged peptide KRT(p)LGARELQTAVR (amino acids 164–177) appeared at 560.3 in the MS scan. The MS/MS spectrum revealed several fragment ions including [M-H₃PO₄+3H]³⁺ at m/z 527.6, and y4, y5, y6, y7, y11²⁺, [y12-18]²⁺. The MS/MS data demonstrated that Thr166 was phosphorylated.

Fig. 5

Hesperadin inhibits TbAUK1 activity and growth of BF and PF trypanosomes. (A) Molecular models of human Aurora A and TbAUK1 were generated based upon the crystal structure of Xenopus Aurora B (2BFY). The top 25 docks to Hesperadin are shown for the models. Each of the bound Hesperadin molecules is represented as a stick figure. (B) Dose response for Hesperadin inhibition of TbAUK1. The left panel shows an autoradiogram with ³²P incorporation into TbH3. The stained Coomassie gel shows that an equivalent amount of TbH3 substrate was loaded onto each gel lane. The right panel shows densitometry of the autoradiograms (n=4; ±SE). (C) Hesperadin inhibits growth of BF and PF cultures. BF or PF were grown in the presence of increasing concentrations of Hesperadin for 24 hr or 48 hr, respectively. The percent growth inhibition was recorded. (D) Time course of cell growth in the presence of increasing concentrations of Hesperadin. BF cultures were treated at time 0 with the indicated concentrations of Hesperadin and cell density was followed for 5 days. The limit of detection was 1×10⁴ cells/ml.

Fig. 6

Treatment with Hesperadin phenocopies RNAi knockdown of TbAUK1. (A) RNAi of TbAUK1 disrupts cell cycle progression in BF. The left panel shows a growth curve of TbAUK1 RNAi cells diluted to 1×10⁵ cells/ml with (+) or without (-) tetracycline. The middle panel is RT-PCR using template RNA isolated from the TbAUK RNAi BF cells after growth for 72 hours with or without tetracycline. Primer pairs were designed to amplify the following: TbAUK1; the upstream gene carbonic anhydrase (CA); the downstream gene dynein heavy chain (DHC); and antisense TbAUK1. Also shown is the PCR control without reverse transcriptase (-RT) and the loading control of ethidium bromide stained total RNA (1 μg). The right panel shows flow cytometry of BF cells at increasing times after RNAi induction. A shift in population to 4C and 8C DNA content is observed. (B) Changes in cell cycle progression induced by RNAi of TbAUK1. The left panels show morphological changes in BF after 24 hr induction with tetracycline. Cells were viewed by DIC, or stained for nuclei (TOTO) and flagella (PFR) (panels a-b). The right panel shows cell cycle progression in the RNAi cells. BF cultures were untreated, or induced with tetracycline for the amount of time indicated. After DAPI staining, cells were evaluated microscopically for their number of nuclei (N) and kinetoplasts (K). Cells designated 1N1K have one nucleus and one kinetoplast. Each time point is the evaluation of at least 200 cells (n=2, ±SE). (C) Changes in cell cycle progression induced by Hesperadin. The left panels show morphological changes in cells treated with 100 nM Hesperadin for 24 hr (panels c-d); The right panel shows cell cycle progression of Hesperadin treated cells. The methods are the same as described in panel B.

Fig. 7

Proliferation of nucleoli following treatment with Hesperadin or depletion of TbAUK1 with RNAi. (A) Nucleoli in Hesperadin treated cells. Cells were treated with 200 nM Hesperadin for the times indicated. The nucleus was stained with DAPI, and nucleoli were labeled with antibody L1C6 and secondary antibodies coupled to Cy3. At least 200 cells were analyzed at each time (n=2; ±SE). (B) Nucleoli in cells depleted of TbAUK1 by RNAi. The number of nucleoli in TbAUK1 RNAi cells was evaluated at different times after induction with tetracycline. At least 200 cells were analyzed at each time (n=2; ±SE). (C) Nucleolar labeling with antibody L1C6. Trypanosomes were left untreated (panel a); were examined after 24 hours of induction with tetracycline (+ Tet) (panels b-d); or examined after 24 hr treatment with 200 nM Hesperadin (panels e-g). With RNAi and Hesperadin, note the disruption in nuclear division leading to swollen, multilobed nuclei and multiple nucleoli. The bars are size markers of 10 μm.