Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors

Abstract

The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei, the proteins that make up the kinetochore are highly divergent; the inner kinetochore comprises at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases-CLK1 (also known as KKT10), CLK2 (also known as KKT19), KKT2 and KKT3. Here, we report the identification and characterization of the amidobenzimidazoles (AB) protein kinase inhibitors that show nanomolar potency against T. brucei bloodstream forms, Leishmania and Trypanosoma cruzi. We performed target deconvolution analysis using a selection of 29 T. brucei mutants that overexpress known essential protein kinases, and identified CLK1 as a primary target. Biochemical studies and the co-crystal structure of CLK1 in complex with AB1 show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with Cys 215 in the ATP-binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell-cycle progression, leading to cell death. This research highlights a unique drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.

Extended Data Fig. 1. High-throughput screening of kinase library and characterization of AB1 effect on T. brucei.

(a) Identification of amidobenzimidazole (AB) series as a scaffold worthy of progression. Single point screen of ten thousand compounds from kinase library were screened against T. b. brucei parasite for growth inhibition at 10 μM. 2264 cpds showed >50% growth inhibition, of which 306 compounds were prioritized based on the cluster analysis and compound availability for 10 point dose response testing. 232 cpds showed <10 μM EC₅₀, further analysis based on physicochemical properties [calculated octanol-water partition coefficient (cLogP) < 4.5; polar surface area (PSA) <110; molecular weight (MW) <500], removal of unfavourable chemical moeities and selectivity index of >10 (cytotoxicity in human hepatocytes HepG2 CC50 / T. b. brucei EC₅₀) lead to identification of amidobenzimidazole series. (b) Time to kill analysis of AB1 compound. Note the concentration and time dependent killing of bloodstream form of T. b. brucei parasites by the AB1 compound. Data shown represent the mean ± SEM (n=3 independent biological replicates). (c) The absolute concentrations (AC_cure) required to achieve sterile cure under in vitro conditions with incubation of compound at various time points. Note that as the time of incubation increases, ACcure reduces. Mean data from two independent biological replicates are shown.

Extended Data Fig. 2. AB1 reduces parasitaemia in a GVR35 strain CNS mouse model of human African trypanosomiasis and pharmacokinetics of AB1.

(a) Whole body in vivo imaging of bioluminescent T. brucei before and after AB1 treatment; T. b. brucei (GVR35-VSL2) –infected mice (Day 21) were orally treated with 50 mg kg^-1 AB1 once-daily for 7 days (n=6 mice, grey) or left untreated (n=3 mice, green); Data represent the mean ± standard deviation of each mice group, and symbols show whole body bioluminescence values for individual mice; mice were euthanized between days 27 and 35. (b) Bioluminescence images of mice showing ventral views of two representative mice from the untreated and AB1-treated groups over the full course of infection. The colour scale indicates bioluminescent radiance in photons/sec/cm²/sr. ND, not detected. Blood parasitemia (in parasites mL^-1, red font below image) and whole mouse total flux (in photons per second, black font above image) values of each animal are shown. The same two representative mice are shown for all time points. (c) Total blood concentration of AB1 in mice at various time points after last dose from the T. b. brucei GVR35 strain CNS mouse model of HAT. At each time point, 3 mice were bled to collect samples, each point represents mean ± standard deviation. (d) Free brain concentrations of AB1 compound in mice calculated by taking into consideration the brain to plasma ratio (0.5), mice plasma protein binding (94%) and rat brain tissue binding (>99%). Each point represents free brain concentrations for n=3 mice and mean ± standard deviation is shown. Note the AB1 concentrations were below T. b. brucei EC₅₀ and AC_cure.

Extended Data Fig. 3. CLK1 target validation and CLK1 over- expression toxicity blockage by AB1.

(a) CLK1 depletion halts cell proliferation. Growth curves of CLK1-depleted bloodstream trypanosomes after RNAi induction with tetracycline (dashed line) is compared with uninduced (solid line) as control. Data represent the mean of two biological replicates. (b) Efficiency of CLK1 RNAi. Upper panel: Endogenous CLK1::12-myc (predicted MW 70.5 kDa) was detected with anti-myc antibody at different points after RNAi induction. EF-1 alpha protein expression was used as the loading control. Lower panel: Mean intensity of the bands were determined from the Western blot in the upper panel. Density signal of CLK1::12-myc was normalised to EF1α for each time point and the uninduced control and relative expression of CLK1::12-myc to uninduced control is shown. Data are representative from one of two independent biological replicates with similar results. (c) Depletion of CLK1 induces a G2/M cell cycle arrest. Flow cytometry analysis of DNA content in CLK1-depleted cells (dark grey) and in the uninduced control (light grey) 4 and 48 h after RNAi induction with 1μg mL^-1 tetracycline. Mean ± SEM (n=3); P values were calculated using a Two-tailed Student’s t-tests comparing G2/M percentage with uninduced control where ** p =2.7 x10^-3 (24 h), *** p =1.6 x10^-4 (48 h). (d) CLK1 overexpression impairs parasite growth. Top: 48 h growth curve of induced TbCLK1 over- expression (OE) compared with uninduced cell line and parental T. b. brucei 2T1 wild-type parasites. Data represent the mean ± standard deviation of three biological replicates where P- values were calculated using a Two-tailed Student’s t-test; *** p= 5.9 x 10^-5. Bottom: CLK1-6-myc overexpression of two clones was assessed by western blot, using an anti-myc antibody. EF-1 alpha protein expression was used as the loading control. (e) Incubation with AB1 impairs toxicity of CLK1 overexpression. Parental 2T1 and CLK1 over- expression (OE) cell lines were pre-incubated or not 18 h with 60 nM of AB1 and AB2 and then incubated with tetracycline during 48 h. Bars show the mean ± SEM (n = 3 biological replicates), and symbols represent the individual parasite counts. *** p = 1.6 x10^-4 compared to induced CLK1 OE preincubated with DMSO. (f) CLK1 target validation using non-toxic concentrations of AB1. 24 h pre-treatment with AB1 non-toxic concentrations (60 nM) reduced the loss of fitness caused by CLK1 overexpression induced by tetracycline. Bars represent the mean ± SEM (n = 4 biological replicates) of the percentage of maximal survival in each condition, and symbols represent the individual replicates. p < 0.001 compared to DMSO control for all the points.

Extended Data Fig. 4. CLK1 and CLK2 protein sequence alignment and CLK1 conservation across trypanosomatids.

(a) Alignment of CLK1/KKT10 and CLK2/KKT19 protein sequence in T. brucei. The protein sequences of CLK1 (Tb927.11.12410) and CLK2 (Tb927.11.12420) were aligned in CLC Genomics Workbench 8 and the consensus graph is shown below the corresponding alignment. Residue Cysteine 215 is shown in blue. (b) Left: CLK1 and CLK2 mRNA levels measured by quantitative RT-PCR (bar graphs) in the 2T1 parental cell line, using GADPH as housekeeping gene. Mean ± SEM (n=3 biological replicates). Right: Protein levels of Ty-YFP-tagged KKT10 and KKT19 were assessed by western blot. Representative of two independent clones is shown. EF-1 alpha protein expression was used as the loading control. (c) The protein sequences of CLK1/KKT10 (Tb927.11.12410) and orthologues from Leishmania mexicana (LmxM.09.0400) and Trypanosoma cruzi (TcCLB.509027.60) were aligned in CLC Genomics Workbench 8. The consensus graph is shown below the corresponding alignment. Conserved residue Cysteine 215 is shown in blue.

Extended Data Fig. 5. Similarity of hEGFR and CLK1 binding sites highlighting potential of AB1 to covalently bind to TbCLK1

(a) Sequence alignment of hEGFR (Uniprot id: P00533), hCLK1 (Uniprot id: P49759) and TbCLK1 show similarity in binding site residues with presence of covalent cysteine (red) in TbCLK1 explaining selectivity over hCLK1. (b) Comparison of ribbon diagram x-ray crystal structure of hCLK1 (PDB: 6FT8), TbCLK1 (PDB: 6Q2A), and hEGFR (5FEQ). The kinase components are coloured and labelled in TbCLK1 (middle panel). Note the conservation of cysteine in the kinase domain in TbCLK1 (C215) and hEGFR (C797), whereas in the same position, hCLK1 has serine 249. (c) An overlay of TbCLK1 with hCLK1 (left) and hEGFR (right) structures.

Extended Data Fig. 6. Covalent binding of AB1 to TbCLK1

(a) Summary of thermal shift assay of TbCLK1 and TbCLK1 C215A proteins incubated with DMSO, AB0 or AB1 compounds. Data represents mean ± standard deviation (n = 3 technical replicates) for ΔTm. Note the significant shift in Tm for AB1 compound bound to CLK1 but not CLK1 C215A. The Tm was calculated by fitting the fluorescence vs. temperature data to the Boltzmann equation and represents the point where half the protein is unfolded. (b) Mass spectrum chromatograms of TbCLK1 (left) and TbCLK1 C215A proteins (right) incubated with DMSO (control), AB1 or AB2 compounds. Y-axis and X-axis represent the counts and deconvoluted Mass (amu) respectively. Calculated differences in base mass and the difference in mass after treatment is shown in the table. Note the shift in mass of 475 Da only for AB1 treatment with TbCLK1 protein, which corresponds to the mass of AB1. No shift in the mass was seen for DMSO or AB2 in both CLK1 and CLK1 C215A protein. Treatment of CLK1 C215A with AB1 also did not result in shift in mass, clearly showing covalent interaction of the Michael acceptor with C215 of TbCLK1 (n=1).

Extended Data Fig. 7. Expression of CLK1 C215A mutant in T. brucei

a) Expression of CLK1 C215A results in a G2/M cell cycle arrest. Induction of recoded CLK1 C215A and C215S mutants were expressed for 24 h with tetracycline and cell cycle distribution was determined by flow cytometry and compared with 2T1 parental cell line. Bars represent the mean ± SEM (n = 3 independent experiments). P values were calculated using a Two-tailed Student’s t-test comparing G2/M phase with parental 2T1 control where *** p = 6.0 x 10^-6. b) Constitutive expression of recoded CLK1 C215S halts CLK1 RNAi growth inhibition. Parasites were induced or not with 1μg mL^-1 tetracycline at 1 x 10⁵ parasites ml^-1 and daily diluted up to 48 h, where ~ 25% reduction in parasite growth was observed. Data represent mean ± SEM (n= 4 technical replicates) showing cumulative cell growth across time. The P values were calculated using a two-tailed Student’s t-test comparing the recoded mutant with parental uninduced control where *** p= 7.9 x 10^-4. A representative Western blot of expression of recoded CLK1 C215S mutant is shown in the right panel. The expression of recoded CLK1 was detected using an anti-HA antibody. EF-1 alpha protein expression was used as the loading control. c) CLK1 C215S mutant impairs the parasite growth effect of 48 h with 5x AB1 EC₅₀ treatment. Growth curve of parasites expressing recoded CLK1 C215S (green) and parental 2T1 cell line (blue) (5 x 10⁵ parasites ml^-1) treated with 5x AB1 EC₅₀ for 48 h. Data represents mean ± SEM (n=3 technical replicates). *** P values were calculated using a Two-tailed Student’s t-test comparing the recoded CLK1 C215S cell line with parental control at each time point, 18 h (p = 3.0 x 10^-6), 24h (p = 1.1 x 10^-4) and 48 h (p = 2.5 x10^-7). d) T. brucei expressing recoded HA-tagged TbCLK1 and C215S mutant were incubated induced or not with 1 μg mL^-1 tetracycline for 24 h and then treated with 5x EC₅₀ AB1 for 6 h. The expression of recoded CLK1 was detected using an anti-HA antibody. EF-1α protein expression was used as the loading control. The size shift of CLK1 is showed with an arrow. Data shown is representative from one of the three biological replicates with similar results.

Extended Data Fig. 8. Cell cycle analysis of synchronized T. brucei after treatment with AB1.

(a) Diagram of 2C (blue) and 4C (orange) cell cycle synchronised bloodstream form T. brucei using Vybrant DyeCycle Violet-based cell sorting. The selected area shows those cells that were gated for sorting. (b - c) After release, 2C and 4C synchronised parasites were treated or not with 5x EC₅₀ AB1. Then, cell samples were collected every hour for DNA flow cytometry analysis of 2C (in blue) and 4C (orange) populations, up to 6 hours. A representative micrograph after 6 hours of treatment is also provided in each condition. Scale bar, 2μm. The data are representative of two biological experiments.

Fig. 1. Pan kinetoplastid activity of amidobenzimidazole (AB) series.

(a) Structure, anti-kinetoplastid parasite activity, cytotoxicity, tyrosine kinase inhibition and in vitro pharmacokinetics of AB0 and AB1. EC_50, and CC₅₀ represent half-maximum growth inhibition concentration. hEGFR: human epidermal growth factor receptor enzyme. BTK: Bruton’s tyrosine kinase. IC₅₀ represents half-maximum enzyme inhibition concentration. All the cellular and biochemical data presented were generated with minimum of n=2 or more independent biological replicates (each biological replicate had n=2 technical replicates). All physicochemical and in vitro pharmacokinetics experiments were carried out with at least n = 4 technical replicates (except for rat microsomal clearance for AB0, which had n =2 technical replicates). (b) Structure activity relationship of amidobenzimidazoles. Each dot represents one compound. Compounds with the Michael acceptor (orange circles) were more potent against T. b. brucei (Tbb pEC₅₀) and had better lipophilic efficiency (clogP, calculated octanol-water partition coefficient) compared to non-Michael acceptor compounds (blue circles). AB0 (dark blue) and AB1 (dark orange). The Tbb pEC₅₀ data are the negative logarithm of 50% growth inhibition of bloodstream form of T. b. brucei (minimum n = 2 biological replicates). The compounds on the left side of the two slanted dotted lines represent lipophilic ligand efficiency (LipE) of 3 and 4. The compounds above two horizontal dotted lines have EC₅₀ of 1000 and 100 nM against bloodstream form T. b. brucei. (c) In vivo activity of AB1 in a haemolymphatic mouse model for human African trypanosomiasis (untreated control group, mice = 4; all the other experimental groups, mice = 6). Each group of mice were infected with T. b. brucei and 3 days post infection, mice were treated with varying concentration of AB1 for 4 days once daily and monitored for relapse in parasitemia over a period of 31 days. Cure plot (Kaplan–Meier plot) showing percentage of animals cured over time are shown. Note the dose dependent activity of AB1, achieving complete cure at 50 mg kg^-1 once daily dose (in violet).

Fig. 2. Identification of CLK1 as the molecular target for the AB series.

(a) Inducible overexpression (OE) of essential protein kinases (PKs) and target deconvolution approach for the AB series. Schematic representation of the experimental workflow: 6-myc OE plasmids, each containing a specific protein kinase tagged with six myc epitopes, were individually transfected into 2T1 bloodstream form T. b. brucei (VSG221 expressing, Tagged, clone 1). Viability of induced individual T. b. brucei OE lines after treatment was assessed by measuring the conversion of resazurin (Alamar blue) to resorufin. (b) AB1 half-maximal effective concentration (EC₅₀) was analysed from twenty-nine essential individual protein kinase over-expression cell lines (dark grey circles). The graph represents EC₅₀ fold change over the parental cell line (T. b. brucei 2T1 cell line, DMSO) (light grey shade box), where overexpression of CLK1 confers resistance to AB1. The grey bar represents the EC₅₀ average of all cell lines over control. Box error bars represent the library mean with 95% of confidence interval of two biological replicates of the library and CLK1 (n = 4 biological replicates). P-value was calculated using two-tailed Student’s t-tests comparing CLK1 OE with parental cell line where *** p-value = 5 x 10^-5. (c) CLK1 over-expression confers resistance to AB1-induced cell cycle arrest. CLK1 over-expression was induced or not with tetracycline for 18 h, and cells then incubated for 6 h with 5x AB1 EC₅₀ (dark grey bars); the 2T1 cell line was a parental control. Cell cycle distribution was determined by flow cytometry. Left: dotted line represents (basal) G2/M untreated average and *** represents a p-value =1.2 x 10^-4 (2T1 untreated vs AB1), p-value = 5.8 x 10^-4 (CLK1 OE uninduced vs AB1), and not signficant (ns) p-value = 0.02 (CLK1 OE induced vs AB1). P-value was calculated using two-tailed Student’s t-test. Error bars represent mean ± SEM of three biological replicates, Right: representative cell cycle profile histogram of cells stained with propidium iodide showing G2/M cell cycle accumulation (arrow). (d) Violin plot of average length (μm) of the nuclei from parasites treated (blue) or not (grey) with AB1 (n=160 2K1N parasites, *** p-value = 3.9 x 10^-26). Dashed line represents the mean value, and dotted lines indicate both quartiles (25^th & 75^th). P-value was calculated using unpaired two-tailed Student’s t-test. Right: Example of a cell stained with DAPI (cyan) from each condition, where N= nucleus, K= Kinetoplast. Scale bar, 2μm. (e) Cell cycle analysis of synchronized T. b. brucei after treatment with AB1. Top: Kinetoplast/nucleus configuration of 2C and 4C synchronized parasites, treated or not with 5x EC₅₀ AB1 was quantified by DAPI staining at the indicated points (n=200 cells). K = kDNA, N = nucleus, D = duplicating kDNA. Bottom: Schematic representation of T. brucei kinetoplast/nucleus configuration through the cell cycle.

Fig. 3. Mechanism of CLK1 inhibition by AB1

(a) Positive correlation between inhibition of recombinant T. brucei CLK1 enzyme (apparent pIC₅₀) and growth inhibition (pEC₅₀) of T. b. brucei bloodstream form parasites with AB series compounds (n=260; Pearson correlation coefficient (r) = 0.68. Presence (orange) or absence (blue) of Michael acceptor pharmacophore is shown, highlighting AB1 (dark orange) and AB0 (dark blue). Black dash line represents equimolar potency and grey dash line represents 10-fold higher potency for CLK1 pIC₅₀ compared to Tbb pEC₅₀. (b)Lack of correlation between inhibition of recombinant human CLK1 (hCLK1) and recombinant T. brucei CLK1 enzyme (apparent pIC₅₀) with AB compound series (n=228; Pearson correlation coefficient (r) = 0.03). Majority of compounds showed > 10-fold selectivity against TbCLK1 compared to hCLK1. Presence (orange) or absence (blue) of Michael acceptor pharmacophore is shown, highlighting AB1 (dark orange) and AB0 (dark blue). Grey dash line represents equimolar potency and black dash line represents 10-fold higher potency for CLK1 pIC₅₀ compared to hCLK1 pIC₅₀. (c) Crystal structure of the T. brucei CLK1 kinase domain (V117-M465) in a covalent thioether bond with AB1 at C215 (PDB: 6Q2A). The CLK1 kinase domain is shown in grey, with a blue αC helix, and the side chains of the DFG motif (DLG in CLK1) shown as sticks. Inhibitor AB1 is shown as ball and sticks with carbon coloured purple, oxygen coloured red and nitrogen coloured blue. A semi-transparent representation of AB1 van der Waals surfaces are shown in magenta. Inset depicts the detailed view of the interactions of AB1. Hydrogen bonds and salt bridge are shown as dotted blue line. MAP-like insertion is showed in orange. (d) Omit 2Fo-Fc map of AB1/C215 at 2.6Å resolution contoured at 2σ. Hydrogen bonds and salt bridge between the AB1 molecule and T. brucei bCLK1 are drawn as blue lines. Covalent bond indicated by yellow arrow.

Fig. 4. CLK1 is the primary target for AB1 in bloodstream form trypanosomes

(a) AB1 binding to CLK1, as probed via differential scanning fluorimetry (DSF). Thermal unfolding of CLK1 is monitored by SYPRO Orange. CLK1 WT (left) and CLK1 C215A (right) recombinant protein in the presence of AB0 (green), AB1 (red) or DMSO (blue) as a reference. Data from one representative experiment, out of three independent experiment with similar results, are shown. Note the shift in Tm (melting temperature of protein) for CLK1 in presence of AB1 compound. (b) Expression of T. brucei CLK1 C215A mutant is resistant to AB1. Dose-response curves of apparent AB1 and staurosporine IC₅₀ of recombinant WT T. brucei CLK1 and C215A mutant. Response to 3-fold serial dilutions of each compound were assessed as described in methods. Each data point represents the mean of two technical replicates. (c) Overexpression of CLK1 C215S mutant confers > 60-fold EC₅₀ shift to AB1. CLK1 C215S mutation (green) was overexpressed in the parental T. b. brucei 2T1 cell line and AB1 half-maximal effective concentration (EC₅₀) was determined after 72 h and compared with T. b. brucei 2T1 WT parental cell line (blue). Data represent mean ± SEM of three independent biological experiments. Inset: Tetracycline inducible over-expression of CLK1-myc was assessed for three clones by western blot, using an anti-myc antibody. EF1α was used as the loading control. (d) Overexpression of CLK1 C215S mutant impairs the parasite growth effect of AB1 treatment. Parasites overexpressing CLK1 C215S (green) and parental T. b. brucei 2T1 cell line (blue) were treated with 5x (line) or 10x AB1 EC₅₀ (dashed line) during 48 h. Mean ± SEM (n=3) is shown. Data represent the mean of the percentage of cell growth relative to time=0 h (1.5 x 10⁴ parasites ml^-1). P values were calculated using a two-tailed Student’s t-test comparing with parental control where *** represent the P-values of 24 h (p-value = 5.4 x 10^-4), 48 h (p-value = 3.4 x 10-4), and 72 h (p-value = 3.3 x 10^-4) after treatment with 5x AB1 EC₅₀, and 24 h (p-value = 9.9 x10^-7), 48 h (p-value = 1.7 x 10^-3), and 72 h (p-value = 1.6 x 10^-3) for 10x AB1 EC₅₀ treatment. (e) Percentage of survival of parasites exposed to 5-150x AB1 EC₅₀ fold range. Parental T. b. brucei 2T1 control (blue) and CLK1 C215S mutant (green) (2 x 10³ parasites ml-) were exposed for 72 h to 5, 15, 50, and 150-fold AB1 EC₅₀. Survival was calculated by using CellTiter-Glo® luminescent cell viability assay. Data represent the mean ± SEM of percentage of survival. (C215S mutant n=3, Parental 2T1 n=4).

Fig. 5. CLK1 inhibition impairs inner kinetochore dynamics.

(a) Localization of kinetochore protein KKT2 after CLK1 inhibition by AB1. Parasites were incubated or not for 24 h with 2x EC₅₀ (upper panel) or 6 h with 5 x EC₅₀ AB1 (lower panel). Representative fluorescence micrographs, showing bloodstream form parasites endogenously expressing N-terminal mNeonGreen (mNG) tagged KKT2. Cells in metaphase and anaphase are shown. Cells were counterstained with DAPI to visualize DNA (cyan). The right panel shows the Nomarsky (DIC) corresponding images. Scale bar, 2μm. Upper right panel shows cell cycle progression after treatment with 2x EC₅₀ AB1 for 72 h. Data are representative from one of three independent biological replicates with similar results. (b) Localization of KKT2 after CLK1 depletion by RNAi. Representative fluorescence micrographs, showing 24 h induction of CLK1 RNAi in bloodstream form parasites endogenously expressing N-terminal mNeonGreen (mNG) labelled KKT2, compared with not induced control cells in metaphase and anaphase are shown. Cells were counterstained with DAPI to visualize DNA (cyan). The right panel shows the Nomarsky (DIC) corresponding images. Scale bar, 2μm. Data are representative from one of three independent biological replicates with similar results.