title: Novel Effects of Lapatinib Revealed in the African Trypanosome by Using Hypothesis-Generating Proteomics and Chemical Biology Strategies.
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.
authors.affiliation: Department of Cellular Biology, Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Guyett
authors.affiliation: Department of Cellular Biology, Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA.
authors.forename: Ranjan
authors.initials: R
authors.lastname: Behera
authors.affiliation: Proteomics Facility, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Ogata
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Pollastri
authors.affiliation: Department of Cellular Biology, Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA mensawil@uga.edu.
authors.forename: Kojo
authors.initials: K
authors.lastname: Mensa-Wilmot
mesh_terms: D002846:Chromatography, Affinity; D004705:Endocytosis / Q000187:drug effects; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D000077341:Lapatinib; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D015800:Protozoan Proteins / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D053719:Tandem Mass Spectrometry; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000378:metabolism / Q000472:pathogenicity*; D014353:Trypanosomiasis, African / Q000469:parasitology*
chemical_list: D010455:Peptides; D015800:Protozoan Proteins; D011799:Quinazolines; D000077341:Lapatinib; D066246:ErbB Receptors
keywords: chemical biology; endocytosis; flagella; kinase inhibitor; paraflagellar rod; phosphoprotein signaling; proteomics