Mechanisms of Action: Physiological Effects

Novel Effects of Lapatinib Revealed in the African Trypanosome by Using Hypothesis-Generating Proteomics and Chemical Biology Strategies

Paul J. Guyett, Ranjan Behera, Yuko Ogata, Michael Pollastri, Kojo Mensa-Wilmot
DOI: 10.1128/AAC.01865-16

ABSTRACT

Human African trypanosomiasis is a neglected tropical disease caused by the protozoan parasite Trypanosoma brucei. Lapatinib, a human epidermal growth factor receptor (EGFR) inhibitor, can cure 25% of trypanosome-infected mice, although the parasite lacks EGFR-like tyrosine kinases. Four trypanosome protein kinases associate with lapatinib, suggesting that the drug may be a multitargeted inhibitor of phosphoprotein signaling in the bloodstream trypanosome. Phosphoprotein signaling pathways in T. brucei have diverged significantly from those in humans. As a first step in the evaluation of the polypharmacology of lapatinib in T. brucei, we performed a proteome-wide phosphopeptide analysis before and after drug addition to cells. Lapatinib caused dephosphorylation of Ser/Thr sites on proteins predicted to be involved in scaffolding, gene expression, and intracellular vesicle trafficking. To explore the perturbation of phosphotyrosine (pTyr)-dependent signaling by lapatinib, proteins in lapatinib-susceptible pTyr complexes were identified by affinity chromatography; they included BILBO-1, MORN, and paraflagellar rod (PFR) proteins PFR1 and PFR2. These data led us to hypothesize that lapatinib disrupts PFR functions and/or endocytosis in the trypanosome. In direct chemical biology tests of these speculations, lapatinib-treated trypanosomes (i) lost segments of the PFR inside the flagellum, (ii) were inhibited in the endocytosis of transferrin, and (iii) changed morphology from long and slender to rounded. Thus, our hypothesis-generating phosphoproteomics strategy predicted novel physiological pathways perturbed by lapatinib, which were verified experimentally. General implications of this workflow for identifying signaling pathways perturbed by drug hits discovered in phenotypic screens are discussed.

This article is dedicated to the memory of Martin John Rogers (1960-2014), who served with passion, extraordinary vision, and dedication as program officer for Tropical Diseases at NIAID. We have lost a warrior and leader. We continue the battle, John.

INTRODUCTION

Human African trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. Current chemotherapies need improvement, and the desired “target product profile” of new therapies include oral administration and multitargeting (1). Lapatinib, a human epidermal growth factor receptor (EGFR) inhibitor (2, 3), was identified as a hit for antitrypanosome drug discovery (4) and subsequently demonstrated to be a lead drug, curing 25% of mice infected with trypanosomes (5). As a promising lead, new analogs of the 4-anilinoquinazoline scaffold in lapatinib are being optimized to improve efficacy and physicochemical properties for HAT drug discovery (6–8).

Although T. brucei lacks EGFR, lapatinib associates with four protein kinases termed T. brucei lapatinib binding protein kinases (TbLBPK1 to -4) (4). Since lapatinib associates with multiple protein kinases, it is likely that the drug perturbs phosphoprotein signaling in T. brucei. Further, the drug's influence on phosphoprotein-dependent processes is likely to be complex, with the drug's polypharmacology leading to multiple effects on trypanosome biology. Even if the function of each TbLBPK was known, it might not be possible to predict the exact effects of lapatinib on the cell because of unknown cross talk between multiple signaling pathways perturbed by the drug.

To discover the direct effects of lapatinib on T. brucei physiology, we adopted a global proteome-wide approach and used hypothesis-generating chemical proteomics to identify proteins in lapatinib-perturbable signaling pathways. Based on the predicted functions of these proteins, we tested the effects of the drug on select aspects of trypanosome biology. Discovery and testing of the biological effects of lapatinib in the trypanosome establish hypothesis-generating proteomics in combination with chemical biology as a valid strategy for pharmacological investigations, especially in cells with evolutionarily divergent signaling pathways.

RESULTS

Hypothesis-generating phosphoproteomics: discovery of proteins in lapatinib-susceptible signaling pathways.Steady-state protein phosphorylation is the equilibrium between phosphorylation by protein kinases and dephosphorylation by protein phosphatases. Lapatinib is a protein kinase inhibitor (9). By perturbing kinase activity, lapatinib likely affects phosphorylation states of downstream effector proteins of TbLBPKs (4). To discover lapatinib-induced proteome-wide changes to phosphoproteins, we used stable incorporation of amino acids in cell culture (SILAC) to quantitate phosphopeptides in dimethyl sulfoxide (DMSO) (control)- versus lapatinib-treated trypanosomes by mass spectrometry. The concentration of lapatinib used (10 μM) was cytostatic after 3 h of exposure (see Fig. S1A in the supplemental material). Thus, changes in phosphorylation do not have a major contribution from dying trypanosomes. Phosphopeptides are reported in Table 1 only if they were observed in all SILAC experiments with a >4-fold change between DMSO and lapatinib treatment (heavy-to-light [H/L] ratio < 0.25). (A complete list of phosphopeptides is available in Tables S1 and S2 in the supplemental material.) As a control, we treated light- and heavy-labeled trypanosomes with DMSO for 1 h and compared phosphopeptide abundance. This control is important because it provides information on the variation in phosphopeptide abundance in the absence of drug and establishes a baseline for calculating changes observed after lapatinib treatment. Representative distributions of heavy-to-light ratios of both sets of SILAC data (DMSO versus lapatinib and the control DMSO versus DMSO) are presented in Fig. 1. Phosphopeptides significantly affected by lapatinib treatment are listed in Table 1 (H/L ratio comparisons of DMSO versus lapatinib or DMSO versus DMSO SILAC experiments with P values of <0.05).

View this table:
TABLE 1

Lapatinib-susceptible phosphopeptidesa

FIG 1
FIG 1

Effects of lapatinib on phosphopeptides. Bloodstream trypanosomes were labeled in light (L)- or heavy (H)-isotope HMI-9 medium (SILAC). Subsequently, light- or heavy-labeled trypanosomes (total, 2 × 108 trypanosomes in 250 ml medium) were treated for 1 h with either DMSO (0.1%) or lapatinib, respectively (red diamonds). In a control experiment, both light- and heavy-labeled trypanosomes were treated with DMSO (0.1%) for 1 h (blue, squares). After treatment, light- and heavy-labeled trypanosomes were mixed, lysed, and trypsinized. Phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC) and identified and quantified by LC-MS/MS. Two biological replicates of each SILAC experiment were analyzed twice by LC-MS/MS as technical replicates (total, four data sets per treatment condition). Each peptide is plotted based on spectral match PEP score versus the relative abundance in the heavy- to light-labeled trypanosomes.

Proteins (whose functions have been predicted reliably from sequence alignments [10] or demonstrated experimentally) with altered phosphorylation could be classified as putative effectors in three general cellular processes: vesicle transport, gene expression, and protein scaffolding. Perturbation of the phosphoregulation of these pathways likely impacts the biology of the trypanosome.

To determine whether lapatinib affects Tyr phosphorylation in the trypanosome, we used a general anti-pTyr antibody to enrich for peptides containing pTyr before and after lapatinib treatment. Immunoaffinity enrichment is required because Tyr phosphorylation is rare and difficult to reliably quantitate in “shotgun” phosphoproteomics experiments. In a Western blot assay developed with P-Tyr-100 (11), trypanosomes treated with lapatinib (whose structure is presented in Fig. 2A) lost signal intensity for pTyr on several proteins, the most prominent of which are marked with asterisks (see lane 2 of Fig. 2B). A control experiment in which α-tubulin in the lysate was detected with anti-α-tubulin antibody showed that similar amounts of protein were loaded per lane in the SDS-polyacrylamide gel (Fig. 2B). We confirmed these observations with immunofluorescence studies at the single-cell level using the P-Tyr-100 antibody. Control cells show pTyr foci, but the lapatinib-treated trypanosomes lost a significant fraction of that signal (Fig. 2C). Together, these data suggest that lapatinib perturbs Tyr phosphorylation in bloodstream trypanosomes.

FIG 2
FIG 2

Lapatinib inhibits tyrosine phosphorylation of proteins in T. brucei. (A) Chemical structure of lapatinib. (B) Western blot detection of tyrosine-phosphorylated proteins using P-Tyr-100 antibody. Bloodstream T. brucei cells in log phase (8 × 105/ml) were concentrated to 107 cells/ml and treated with either DMSO (control) or lapatinib (10 μM) in HMI-9 medium for 2 h at 37°C. Cells were pelleted, lysed in 1× SDS-PAGE sample buffer, and proteins were resolved on 10% polyacrylamide gel. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane for Western blotting. Lanes 1 and 3 are DMSO-treated samples, and lanes 2 and 4 are lapatinib-treated samples. Western blots against pTyr are shown in lanes 1 and 2, and Coomassie blue-stained total protein load control (of a gel run in parallel) is shown in lanes 3 and 4. The anti-α-tubulin blot is a control for the total protein loading. *, decrease in the protein level after treatment with lapatinib; ◆, protein that remains unaffected. (C) Single-cell immunofluorescence assay: effects of lapatinib on tyrosine-phosphorylated proteins. T. brucei cells were treated as described for panel B and fixed in 4% paraformaldehyde, and pTyr on proteins was detected with anti-pTyr antibody P-Tyr-100. DAPI staining of the nucleus (n) and the kinetoplast (k) (mitochondrial DNA) is shown.

Identifying pTyr-regulated pathways affected by lapatinib will help our understanding of the drug's antitrypanosomal activity in vitro or in a mouse model of HAT (5). Since pTyr serves as a docking site for effector proteins (12), we reasoned that polypeptides in complex with Tyr-phosphorylated proteins might provide some insight into processes regulated by that posttranslational modification. We used anti-pTyr antibody to pull down Tyr-phosphorylated proteins and their strongly associated polypeptides. Proteins whose binding to the anti-pTyr antibody column was reduced 2-fold (or more) when cells were pretreated with lapatinib were likely involved in lapatinib-susceptible pTyr signaling pathways.

Equivalent amounts of total protein were loaded onto anti-pTyr affinity or control Sepharose CL4B matrices. After the columns were washed with buffer containing 1 M KCl to reduce nonspecific protein binding, proteins were eluted sequentially with Tyrphostin A47 and then phenyl phosphate, which are Tyr and pTyr mimics (13, 14), respectively. Finally, the columns were eluted with SDS (5%). Proteins in each eluate were separated by SDS-PAGE, silver-stained, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS) (15–17). The relative amounts of proteins between control and lapatinib-treated cells were quantified by spectral counting (18, 19) (see Tables S5 and S6 in the supplemental material). These experiments were repeated three times with independent biological samples; proteins are reported only if their effects were statistically significant across all three experiments (P < 0.05).

It is necessary to make a technical note about the sequential polypeptide elution strategy employed above. The use of Tyrphostin A47 to elute proteins from a P-Tyr-100 column is novel. It is justified by the similarity of the structure of Tyrphostin A47 to that of Tyr (not to pTyr). Our initial attempts to elute proteins from the anti-pTyr antibody employed only phenyl phosphate, but the pattern of eluted proteins was not reproducible until a Tyrphostin A47 elution was performed ahead of phenyl phosphate elution.

Class I polypeptides.Similar amounts of class I polypeptide proteins (see Table S3 in the supplemental material) were detected on the Sepharose CL4B and the anti-pTyr antibody columns. We infer that binding of these proteins to the columns is not dependent on Tyr phosphorylation.

Class II polypeptides.Class II polypeptides (see Table S4 in the supplemental material) were detected in greater amounts (i.e., more than twice the number of spectral counts) on the anti-pTyr antibody column than on Sepharose CL4B. Pretreatment of T. brucei with lapatinib before P-Tyr-100 affinity chromatography did not reduce the quantity of proteins detected on the anti-pTyr antibody column. Class II proteins (Table S4) are therefore not in a lapatinib-inhibitable pTyr pathway.

Class III polypeptides.For class III polypeptides (Table 2; Tables S5 and S6), a greater fraction bound to the anti-pTyr antibody column than to the Sepharose CL4B column. More importantly, when cells were pretreated with lapatinib, the amount of the protein detected on the anti-pTyr antibody column decreased at least 2-fold (P < 0.05). We conclude that class III proteins are components of lapatinib-susceptible pTyr-signaling pathways (Table 2).

View this table:
TABLE 2

Proteins in lapatinib-susceptible phosphotyrosine pathways (class III proteins)a

Predictions of lapatinib's biological effects from hypothesis-generating proteomics.Taken together, the phosphoproteomic analyses suggest that two essential biological processes may be impacted by lapatinib treatment: (i) flagellum function and/or morphogenesis, because it is dependent on paraflagellar rod (PFR) proteins (20, 21); and (ii) endocytosis, because BILBO-1 (22) and MORN protein (23) are important for maintaining the flagellar pocket in trypanosomes and Rabs regulate endocytosis (24). Could lapatinib's effect on these proteins (Tables 1 and 2) lead to the drug's regulation of PFR function/properties and/or endocytosis in T. brucei? We addressed these questions directly with chemical biology strategies.

PFR structure and cell morphology are altered after lapatinib treatment of trypanosomes.To test if the flagellum, and possibly the paraflagellar rod, was affected after lapatinib treatment (as suggested by pTyr affinity chromatography results [Table 2]), we used an antibody against PFR-A (L8C4) (25) in an immunofluorescence study (26, 27). In untreated cells, the flagellum lay along the length of the long slender trypanosome (Fig. 3A). In “rounded” cells detected after 3-h lapatinib treatment (Fig. 3A), PFR-A was found circumscribing the cell periphery (Fig. 3A), indicating that the flagellum wraps around the cell.

FIG 3
FIG 3

Flagellum topology is altered by lapatinib treatment of T. brucei. (A) Indirect immunofluorescence on DMSO (control)- or 10 μM lapatinib-treated T. brucei cells (5 × 105/ml) using L8C4 (anti-PFR 2 mouse MAb). Cells were treated for 3 h prior to fixation in paraformaldehyde and permeabilization (1% Triton X-100). A secondary anti-mouse antibody conjugated to Alexa Fluor 594 (red) was used. DAPI staining (blue) indicates the positions of nucleus (n) and kinetoplast (k). (B) Scanning electron micrographs (SEM) of control and lapatinib-treated T. brucei. (C) Transmission electron micrographs (TEM) of control and lapatinib-treated T. brucei cells. Paraflagellar rod indicated with arrowhead.

The flagellum was examined in greater detail by electron microscopy. By scanning electron microscopy (SEM), the flagellum appeared wrapped, like a corkscrew, around the rounded cell body of lapatinib-treated trypanosomes (Fig. 3B), while control cells retained the elongated path of the flagellum along the long slender cell. Some lapatinib-treated trypanosomes appeared to resorb their flagella (Fig. 3B, bottom right panel). To determine whether the flagellum was resorbed, we examined sections of trypanosomes by transmission electron microscopy (TEM). In all images, the flagellum had an intact membrane and was separate from the cell body (Fig. 3C); hence, the flagellum remains outside the cell body. Upon closer investigation of the flagellum cross sections, trypanosomes treated with lapatinib lost PFR structure (30% of 61 cross sections), whereas all flagella from control cells had prominent intact PFRs (n = 25) (Fig. 3C, arrowheads). We infer that there is perturbation of the PFR complex, with some regions of the flagellum completely devoid of the PFR (although PFR proteins may be retained inside the flagella, as observed in the immunofluorescence assays [IFAs]) after lapatinib treatment.

Lapatinib's effect on endocytosis of Tf.To test if lapatinib inhibits endocytosis, as predicted above, we used transferrin (Tf) as cargo. Lapatinib inhibited endocytosis of Tf (Fig. 4A): the drug (2 μM) blocked 60% of Tf uptake (determined from a plot of the median fluorescence intensity of data from Fig. 4A) (Fig. 4B). Lapatinib inhibition of endocytosis is rapid, occurring within 15 min of exposure to the drug, and is the earliest measured effect of lapatinib on the trypanosome. After 15 min of exposure, the drug-treated cells had a normal “long slender” shape (see Fig. S2 in the supplemental material) and were not permeable to propidium iodide (PI), which accumulates in dead cells (data used in interpretation of flow data; see Materials and Methods) (28).

FIG 4
FIG 4

Lapatinib inhibits transferrin endocytosis. Trypanosomes (5 × 105/ml) were incubated with transferrin-Alexa Fluor 488 (Tf-Alexa 488) conjugate and various concentrations of lapatinib (there was no preincubation of trypanosomes with the drug) for 15 min. Propidium iodide (PI) was added as described in Materials and Methods. The Tf-Alexa 488 and PI fluorescence of cellular events was determined by flow cytometry. (A) Representative plot of trypanosome Tf-Alexa 488 fluorescence in the presence of lapatinib (dashed line) or DMSO control (solid line). (B) Quantification of endocytosed Tf. Median Tf-Alexa 488 fluorescence of trypanosomes in the presence of 0 μM (DMSO only), 1 μM, 2 μM, or 3 μM lapatinib. Error bar, 1 standard deviation in data from three independent experiments.

DISCUSSION

A major goal of this work was to evaluate the pharmacological basis of lapatinib's trypanocidal effects (4, 5). In human cells, lapatinib inhibits EGFR (2, 3), but it also binds to other protein kinases (29), and it associates with Pgp ATPase (30) as well as ABCB1 transporters (31). There are no EGFR-like protein kinases in the trypanosome. However, four trypanosome protein kinases associate with lapatinib, namely, tousled-like kinase (Tb427.04.5180), casein kinase 1.2 (Tb427.05.800), an uncharacterized protein kinase (Tb427.03.1570), and glycogen synthase kinase (Tb427.10.13780) (4). With attributes of a multitargeted kinase inhibitor, as well as potentially inhibiting other ATPases (4), lapatinib is expected to have pleiotropic effects on trypanosome physiology (32). Determining the function of each target kinase individually may not provide a complete understanding of the polypharmacology of lapatinib because pathways controlled by kinases may intersect in unexpected ways. Since lapatinib is likely to act, at least in part, through perturbation of phosphoprotein homeostasis because it associates with multiple trypanosome protein kinases (4), we measured the drug's direct effect on the phosphoproteome of bloodstream form (BSF) trypanosomes.

Our hypothesis-generating proteomics workflow led to the idea that lapatinib affects flagellum biology, since several PFR proteins were affected (Table 2). These data are consistent with the work of Ferguson and colleagues, who found foci of Tyr-phosphorylation along the flagellum of insect stage T. brucei (33). The PFR is unique to the trypanosomatids and Euglena (34). In T. brucei, the importance of phosphoregulation of PFR proteins, of which there are over 20 (35), is not known. Treatment of trypanosomes with lapatinib correlated with the loss of pTyr on some PFR proteins (Table 2) and compromised the integrity of the PFR (Fig. 3C) as well as the topology of the flagellum (Fig. 3B and C). This phenotype is not without precedent, as knockdown of PFR2 or PFR1 results in loss of PFR observed in TEM cross sections (20). We speculate that dephosphorylation of PFR2 (Table 2) and/or other PFR proteins (Table 2) triggers disassembly of the PFR in regions of the flagellum (Fig. 3C) or incomplete assembly of new PFR during the emergence of a new flagellum. The result of either scenario is that the flagellum structure is altered, which is known to decrease the fitness of trypanosomes in culture and in the mouse model of HAT (36). Based on these observations, we propose that PFR phosphorylation regulates flagellum morphogenesis.

A hint that lapatinib might regulate endocytosis came from the drug causing dephosphorylation of BILBO-1, kinesins, and a Rab (Tables 1 and 2). In trypanosomes, endocytosis occurs at the flagellar pocket, an invagination of the plasma membrane from which the flagellum emerges. BILBO-1 localizes to the neck of the flagellar pocket and affects endocytosis (22). Trypanosome Rabs are also essential for endocytosis (37). Thus, altered phosphorylation of BILBO-1 and/or Rabs may impact transferrin endocytosis (Fig. 4; Fig. S2). This work is not the first evidence that a tyrosine kinase inhibitor affects endocytosis in T. brucei. The pantyrosine kinase inhibitor, Tyrphostin A47, also inhibits transferrin endocytosis (38). Future work may shed light on the importance of phosphorylation in the biological functions of BILBO-1, Rabs, and kinesins.

There was no obvious candidate in the phosphoproteome data (Table 1) whose “loss of function” could explain the altered trypanosome morphology (from long and slender to rounded) (Fig. 2 and 3) after exposure to lapatinib. Trypanosome rounding has been observed in knockdown of proteins that are also involved in endocytosis (39), protein synthesis (40), and cell cycle (41, 42). Trypanosomes have a subpellicular microtubule array (corset) that would need to be reorganized during cell rounding. After lapatinib treatment, the cells become rounded and lose portions of PFR, but the subpellicular microtubules appear intact (Fig. 3C). Future genetic studies of the putative effectors of lapatinib action (Table 1) may shed light on new proteins involved in the cytoskeletal rearrangements needed to transform the trypanosome shape as described in the work presented here.

Undoubtedly, some effects of lapatinib (e.g., inhibition of endocytosis, flagellum topology changes) may be explained by its action on signaling pathways controlled by TbLBPKs (4). For example, a knockdown of TbGSK3β inhibits endocytosis of Tf (43), and trypanosome rounding was observed after RNA interference (RNAi) of TbTLK-1 (41). New studies will be needed to confirm that phosphosignaling pathways downregulated by lapatinib in trypanosomes are a hybrid of those observed when all four TbLBPKs are knocked down genetically.

The current work was performed to better understand how lapatinib decreases fitness of trypanosomes in vitro (4) and during drug treatment of trypanosome-infected mice (5). In our previous study, lapatinib cured 25% of infected mice (5). We presume that the drug's failure to cure the other 75% of mice is a result of its poor pharmacokinetics in mice specifically with regard to controlling the proliferation of trypanosomes. Against bloodstream form T. brucei, the GI50 (concentration of drug causing 50% reduction in growth) of lapatinib is 1.5 μM (4). We did not determine the concentration of lapatinib in plasma in mice that was used in our previous study, in which the drug extended life but did not cure the disease. In humans, the drug accumulates in tissues to a greater extent than in blood, where the concentration is 2 μM (44). Thus, it is possible that lapatinib failed to cure mice of trypanosomiasis because its level in blood did not exceed a threshold (e.g., 10-fold higher than the in vitro-determined GI50) needed for efficacy in a mouse model of HAT. We have an ongoing effort to improve performance of the lapatinib scaffold against T. brucei. We have synthesized analogs that are more potent (i.e., have nanomolar GI50) against T. brucei (6–8), and we will soon test these new hits in the mouse model of HAT.

In considering future lead optimization, we note that lapatinib was optimized against a human EGFR (45) instead of a trypanosome enzyme. Further, we note that the 4-anilinoquinazoline scaffold of lapatinib is present in other marketed drugs (e.g., gefitinib, erlotinib). Considering these facts, we have initiated projects to improve potency of the 4-anilinoquinazole scaffold and its derivatives against trypanosomes with great success (6–8). The next stage of the drug development process, which is ongoing, involves improvement of pharmacokinetic properties as well as adsorption, distribution, metabolism, and excretion (ADME) in vertebrate animals, for example, increasing central nervous system (CNS) penetrance.

In summary, we described an unbiased approach to explore the pharmacology of lapatinib in the African trypanosome. Proteomic analyses led to the hypothesis that lapatinib affects flagellum biology and endocytosis. Chemical biology experiments confirmed that lapatinib treatment destabilizes the PFR structure and inhibits endocytosis of transferrin in bloodstream T. brucei. These two biological processes are essential for the parasite's viability, and their perturbation can explain, at least in part, lapatinib's ability to kill trypanosomes (4, 5). This hypothesis-generating proteomics and chemical biology strategy could find general use in understanding the mode of action of other antiparasite hits discovered in phenotypic screens (46, 47).

MATERIALS AND METHODS

Parasites and cell culture.Bloodstream form Trypanosoma brucei (strain CA427) was obtained from C. C. Wang (University of California, San Francisco) and routinely cultured in HMI-9 medium (48) containing 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 10% Serum Plus (SAFC Biosciences, Lenexa, KS), and 1% antibiotics-antimycotic solution (Cellgro, Manassas, VA) at 37°C, 5% CO2.

Materials.Lapatinib (GW572016 ) was a gift from GlaxoSmithKline (Durham, NC), and Tyrphostin A47 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Antibodies.P-Tyr-100 (antiphosphotyrosine mouse monoclonal antibody [MAb]) and immobilized P-Tyr-100 were from Cell Signaling Technology, Inc. (Danvers, MA). The antibody 12G10 (anti-α-tubulin mouse monoclonal antibody) was from the Developmental Studies Hybridoma Bank (University of Iowa). L8C4 (anti-PFR2 mouse monoclonal antibody) was a gift from K. Gull (University of Oxford) (49).

SILAC phosphopeptide enrichment and LC-MS/MS analysis.Bloodstream form trypanosomes were cultured for at least 5 days (17 doublings) in HMI-9 medium modified for SILAC: Iscove's modified Dulbecco's medium (IMDM) depleted of Lys and Arg was used and supplemented either with l-Arg (120 μM) and l-Lys (240 μM) (“light”) or with 13C6-l-Arg (120 μM) and 2H4-l-Lys (240 μM) (“heavy”). Heavy- or light-labeled BSF cells (log phase, 2 × 108 total trypanosomes in 250 ml medium) were treated with either 10 μM lapatinib or the equivalent volume of DMSO, respectively, for 1 h at 37°C. After treatment, the cells were immediately transferred to ice and washed with PBS-G (phosphate-buffered saline supplemented with 10 mM glucose) and 1 mM Na orthovanadate. The light- and heavy-labeled cells were then combined, lysed by sonication in 50 mM HEPES (pH 7.6)–8 M urea–1 mM Na orthovanadate, and subsequently alkylated with 9 mM iodoacetamide for 30 min. The lysate was then diluted 5-fold with 50 mM HEPES (pH 7.6) and 1 mM Na orthovanadate (final urea concentration, 1.6 M), and the protein was digested with trypsin immobilized on agarose beads for 48 h. After collecting the beads by centrifugation, the peptide supernatant was diluted 10-fold with 0.1% trifluoroacetic acid (TFA) and loaded onto a Sep-Pak C18 column. The peptides were eluted with a step gradient of 1%, 25%, and 50% acetonitrile and dried by air stream. Phosphopeptides were enriched by FeCl3-charged metal affinity chromatography (IMAC) made in-house (Ni-nitrilotriacetic acid [Ni-NTA] was stripped of nickel by washing with EDTA [100 mM] and then charged with FeCl3 just prior to use). Briefly, peptide samples were resuspended in 80% acetonitrile–0.1% TFA and loaded onto FeCl3-charged IMAC resin (10-μl bed volume). The resin was washed three times with 150 μl of 80% acetonitrile in 0.1% TFA and then subjected to a final wash of 1% TFA (150 μl). The peptides were eluted twice (3 min each) with 150 μl of 500 mM potassium phosphate (pH 7). The samples were then desalted using ZipTip C18 (Millipore Corporation) before MS analysis.

Phosphopeptide samples were divided and run as two technical replicates for one (LC-MS/MS) analysis on an Easy-nLC 1000 (Thermo Scientific) coupled to an Orbitrap Fusion mass spectrometer (Thermo Scientific). The LC system consisted of a fused-silica nanospray needle (PicoTip emitter, 75-μm inner diameter [ID]; New Objective) packed in-house with 40 cm of Magic C18 AQ 100-Å reverse-phase medium (Michrom Bioresources Inc.). The peptide sample was diluted in 7 μl of 2% acetonitrile and 0.1% formic acid in water, and 5 μl of the mixture was loaded onto the column and separated using a two-mobile-phase system consisting of 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B). The chromatographic separation was achieved over a 103-min gradient from 2% to 50% B (3 to 27% B for 90 min, 27 to 50% B for 8 min, and 50% B for 5 min) at a flow rate of 300 nl/min. The mass spectrometer was operated in a data-dependent MS/MS mode over the m/z range of 400 to 1,500. The mass resolution was set to 120,000. The cycle time was set to 2 s, and the most abundant ions from the precursor scan were selected for MS/MS analysis using 28% normalized higher-energy collisional dissociation (HCD) collision energy and analyzed with an ion trap. Selected ions were dynamically excluded for 30 s.

Technical replicates were combined and analyzed together using Proteome Discoverer 2.0 (Thermo Scientific). The data were searched using SEAQUEST (50) against T. brucei protein database v. 4.2 (tritrypdb.org, [10]), which included additional common contaminants. Trypsin was set as the enzyme with maximum missed cleavages set to 2. The precursor ion tolerance was set to 10 ppm, and the fragment ion tolerance was set to 0.6 Da. Variable modifications were set to methionine oxidation (+15.995 Da), 6 × 13C (+6.020 Da) on arginine, 4 × 2H (+4.025) on lysine, and phosphorylation (+79.966) on serine, threonine and tyrosine. The search results were run through Percolator (51) for scoring. The results were filtered for peptides identified with a false-discovery rate of <0.05. Phosphorylation sites were evaluated and probability values were calculated using phosphoRS v. 3.1 (52). Phosphorylation sites were considered real if the PhosphoRS site probability was greater than 80%. Peak abundance of light and heavy isotope peptides were determined as an average of the two technical replicates. The ratio of heavy to light peak abundance was determined for each biological sample.

Antiphosphotyrosine affinity chromatography and LC-MS/MS analysis.BSF T. brucei cells (108) were resuspended at a density of 5 × 105/ml in HMI-9 medium and treated with either DMSO (solvent) or lapatinib (10 μM) for 3 h. Cells were harvested, washed twice in ice-cold phosphate-buffered saline (PBS) containing 1% glucose (PBS-G), and lysed in 1 ml lysis buffer (20 mM Tris-HCl [pH 7.4], 60 mM MgCl2, 60 mM KCl, 1 mM dithiothreitol [DTT], 1% Triton X-100, 1 mM sodium vanadate) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF], 2 μg/ml aprotinin, 5 μg/mM leupeptin, 37 μg/ml Nα-Tosyl-Lys-chloromethylketone]TLCK], 2 μM FMKO24). The protein concentration in the cell lysate from each treatment was determined by bicinchoninic acid (BCA) reagent (Pierce), and an equal amount of protein (∼350 μg) was incubated with 24 μl of either Sepharose CL4B (control) or immobilized antiphosphotyrosine antibody (Sepharose P-Tyr-100) column for 4 h at 4°C. The beads were recovered (13,000 × g for 10 min, 4°C) and washed thrice (5 min each) in 500 μl lysis buffer containing 1 M KCl. Proteins were eluted sequentially from each column three times with 50 μl of (i) Tyrphostin A47 (200 μM) in PBS or (ii) phenyl phosphate (200 mM) in PBS and pooled separately (total volume, 150 μl). Between elutions with different solutions, the column was washed with 500 μl lysis buffer to avoid carryover of the eluted proteins into subsequent elutions. Samples were kept on ice during the entire procedure.

The pooled eluates were concentrated by 6% trichloroacetic acid (TCA) precipitation and resuspended in 1× SDS sample buffer. Proteins were separated by SDS-PAGE (on a 10% minigel) and silver stained (Pierce). Each lane of SDS-polyacrylamide gel was cut into five pieces, each of which was further cleaved into 4-mm2 pieces, destained with a silver-stain-destaining solution from a kit (Invitrogen Corporation), and dehydrated with acetonitrile for trypsin digestion.

Proteins were reduced with dithiothreitol (10 mM) in ammonium bicarbonate (100 mM) at 55°C for 45 min and alkylated with iodoacetamide (50 mM) in ammonium bicarbonate (100 mM) for 30 min at room temperature. After dehydration with acetonitrile and rehydration with ammonium bicarbonate twice, proteins were digested overnight with trypsin (5 ng/μl; Promega Corporation) in ammonium bicarbonate (50 mM) at 37°C. Peptides were extracted with formic acid (5% [vol/vol] in water) for 30 min and then with acetonitrile. Extracts were pooled and dried in a SpeedVac, and peptides were purified with ZipTip C18 chromatography (Millipore Corporation).

LC-MS/MS runs were identical to those described above for the phosphopeptide LC-MS/MS analysis. Raw MS/MS data were submitted to the Computational Proteomics Analysis System (CPAS) (53), a Web-based system built on the LabKey Server v11.2 and searched using the X! Tandem search engine (2009.10.01.1) against T. brucei protein database v. 4.1 (from TritrypDB.org), which included additional common contaminants such as human keratin. The database contained 8,614 protein entries, including contaminants. The following were considered variable modifications: carbamidomethylation of cysteine as a fixed modification, phosphorylation of serine, threonine, tyrosine, loss of water or ammonia from terminal glutamic acid or glutamine, respectively, and oxidation of methionine. The mass tolerances were set to ±2 Da and ±0.5 Da for precursor and fragment ions, respectively. The enzyme was set to trypsin, and up to 2 missed cleavages were permitted. The search output files were analyzed and validated by ProteinProphet (PeptideProphet probability ≥ 0.9) (54, 55).

Anti-pTyr Western blotting.Late-log-phase trypanosomes were resuspended in HMI-9 at 107/ml and treated with DMSO (control) or lapatinib (10 μM) for 2 h at 37°C and 5% CO2. Lapatinib-treated and control cells were washed twice in BBS-G (bicine-buffered saline supplemented with 10 mM glucose) and lysed in 2× SDS-PAGE sample buffer. Total proteins (7.5 × 106 cell equivalents per lane) were resolved by 10% SDS-PAGE and transferred to Immobilon P membrane using a semidry electrophoretic transfer cell (56). The membrane was blocked for an hour at room temperature with Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% (wt/vol) nonfat dry milk (blocking buffer) and then incubated overnight at 4°C with P-Tyr-100 antibody (1:500 dilution). The membrane was then washed (four times, 5 min each) with TBST, and the alkaline phosphatase-conjugated goat anti-mouse secondary antibody was applied (1:1,000 dilution in blocking buffer) for 1 h at room temperature, followed by color development with 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (BCIP-NBT). In a parallel experiment, alpha-tubulin in T. brucei was detected with 12G10 antibody (1:10,000 dilution) as a loading control (2.5 × 106 cell equivalents per lane).

Immunofluorescence microscopy.Late-log-phase parasites were treated with lapatinib (10 μM) or DMSO for 2 h at 37°C and 5% CO2. The cells were washed twice in 1 ml of PBS-G (pH 7.4) and fixed with 4% paraformaldehyde in PBS on ice for 1 h. The fixed trypanosomes were adhered to poly-l-lysine-coated coverslips for 30 min and permeabilized in 0.1% Triton X-100 for 25 min at room temperature. The cells were then washed once in PBS and then blocked with 1% bovine serum albumin (BSA) in PBS at room temperature for 1 h. After blocking, cells were incubated for 1 h at room temperature with either P-Tyr-100 (1:400 dilution in blocking buffer) or L8C4 (undiluted) primary antibody. The cells were washed three times with 500 μl PBS (5 min each) and incubated with Alexa Fluor 594- or Alexa Fluor 488-coupled goat anti-mouse or goat anti-rat secondary antibody (1:1,000 dilution in blocking buffer) for 1 h at room temperature. In one set of controls, fixed cells were incubated with the secondary antibodies only. Following antibody incubation, cells were washed five times in PBS and mounted on glass slides with VectaShield containing DAPI (4′,6-diamidino-2-phenylindole). Differential interference contrast (DIC) and fluorescence images were captured and analyzed using an Axio Observer Z1 (inverted fluorescence microscope) fitted with an AxioCam MRm operated by AxioVision 4.6 software.

Scanning electron microscopy.Trypanosomes were harvested in late log phase, resuspended to 5 × 105/ml, and treated with either DMSO (control) or lapatinib (10 μM) for 2 h at 37°C and 5% CO2. The cells were fixed in 2% glutaraldehyde at room temperature for 1 h, washed twice in 1 ml PBS, and transferred to poly-l-lysine-coated coverslips as described earlier. Cells on the coverslips were fixed with 1% osmium tetraoxide for 30 min and dehydrated in increasing concentrations of ethanol (25% to 100%; 5 min each) at room temperature. The samples were dried at critical points with a Tousimis Critical Point Dryer (Samdri-780 A) and sputter coated (gold) with the SPI Module Sputter Coater using standard protocols. Using a Zeiss 1450EP variable-pressure scanning electron microscope, samples were viewed and images captured.

Transmission electron microscopy.After treatment of T. brucei with DMSO or drug (the same treatment as described above for SEM), cells were fixed in 2% glutaraldehyde in PBS for 1 h at 4°C. After washing excess glutaraldehyde with buffer, the samples were then fixed for 1 h at 4°C in 1% OsO4 in PBS. They were then washed once in buffer for 10 min and then subjected to two washes in water to remove excess salts before dehydration in an ethanol series. Once in 100% ethanol, the samples were transitioned into propylene oxide and then incrementally infiltrated with EmBed 812 resin (EMS, Hatfield, PA). The sample was placed in fresh 100% resin and spun to the tip of a 0.5-μl microcentrifuge tube and placed in a 60°C oven overnight to polymerize. The hardened blocks were trimmed and sectioned on an RMC MTX ultramicrotome (RMC/Boekeler, Tucson, AZ) to a thickness of approximately 50 nm. Sections were then poststained with uranyl acetate and lead citrate. The resulting sections were observed on an FEI Tecnai20 TEM (FEI, Inc., Hillborough, OR) operated at 200 KeV, and images were captured digitally with an AMT camera (AMT, Woburn, MA).

Transferrin endocytosis assays.BSF T. brucei cells (5 × 105) cultured in HMI-9 medium were washed at room temperature with 1 ml HMI-9 containing 1% serum and various concentrations of lapatinib (delivered in 1 μl of DMSO) or DMSO alone (1 μl). The cells were resuspended in 1 ml of HMI-9 (1% serum) containing 25 μg/ml transferrin-Alexa Fluor 488 conjugate (Invitrogen, Eugene, OR) and the specified lapatinib concentration (or DMSO). Trypanosomes were incubated for 15 min at 37°C, 5% CO2. Separately, cells (5 × 105) were treated with digitonin (1.5 μM) for 5 min at 37°C, 5% CO2 (permeabilized cell control). A control aliquot of medium and transferrin without cells was also prepared (no cell control). All samples were transferred to ice. For samples analyzed by microscopy, the cells were pelleted (2,000 × g, 5 min, 4°C), washed once with PBS-G, and then fixed in 4% paraformaldehyde at 4°C for 1 h. Fixed cells were adhered to poly-l-Lys-coated coverslips, counterstained with DAPI (1.5 μM) in VectaShield, and mounted on slides. Images were captured using a DeltaVision inverted fluorescence microscope and the SoftWorx Explorer software suite. For samples analyzed by flow cytometry, the cells were pelleted (2,000 × g, 5 min, 4°C), washed once with 1 ml of cold PBS-G, and resuspended in 500 μl of cold binding buffer (10 mM HEPES [pH 7.6], 140 mM NaCl, 2.5 mM CaCl2, 10 mM d-glucose) containing propidium iodide (PI) (3 μM). The samples were incubated with PI for 15 min (on ice) and then analyzed by flow cytometry (CyAn ADP Analyzer; Beckman Coulter) with the following settings: for Tf-Alexa Fluor 488, excitation, 488 laser; emission, 530/40 filter; for PI, excitation, 488 laser; emission, 613/20 filter. Data obtained from the cells gated by forward and side scatter were analyzed in FlowJo software (Tree Star, Ashland, OR). Median Tf-Alexa Fluor 488 fluorescence was determined for events with similar forward scatter versus side scatter profiles that were distinguished from the “no cell” control background noise and were not permeable to propidium iodide.

ACKNOWLEDGMENTS

This work was supported by funds from the NIAID (R56AI099476) to K.M.-W. and M.P. The Fusion Orbitrap mass spectrometer was purchased with a grant from the M.J. Murdock Charitable Trust.

Author contributions: P.J.G. and R.B. performed biological experiments and analyzed results. Y.O. ran and analyzed mass spectrometry samples. P.J.G., M.P., and K.M.-W. interpreted results. All authors contributed to the writing of the manuscript.

FOOTNOTES

    • Received 26 August 2016.
    • Returned for modification 10 October 2016.
    • Accepted 18 November 2016.
    • Accepted manuscript posted online 21 November 2016.

  • Supplemental material for this article may be found at https://doi.org/10.1128/AAC.01865-16 .

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KEYWORDS

proteomics
quinazolines
Trypanosoma brucei brucei
Trypanosomiasis, African
phosphoprotein signaling
proteomics
paraflagellar rod
flagella
endocytosis
chemical biology
kinase inhibitor

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