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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.

SUBMITTER: Guyett PJ

PROVIDER: S-EPMC5278746 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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

Guyett Paul J PJ Behera Ranjan R Ogata Yuko Y Pollastri Michael M Mensa-Wilmot Kojo K

Antimicrobial agents and chemotherapy 20170124 2

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. ...[more]

PMID: 27872081

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Project description:Human African trypanosomiasis is caused by the eukaryotic microbe Trypanosoma brucei. To discover new drugs against the disease, one may use drugs in the clinic for other indications whose chemical scaffolds can be optimized via a medicinal chemistry campaign to achieve greater potency against the trypanosome. Towards this goal, we tested inhibitors of human EGFR and/or VEGFR as possible anti-trypanosome compounds. The 4-anilinoquinazolines canertinib and lapatinib, and the pyrrolopyrimidine AEE788 killed bloodstream T. brucei in vitro with GI(50) in the low micromolar range. Curiously, the genome of T. brucei does not encode EGFR or VEGFR, indicating that the drugs recognize alternate proteins. To discover these novel targets, a trypanosome lysate was adsorbed to an ATP-sepharose matrix and washed with a high salt solution followed by nicotinamide adenine dinucleotide (NAD(+)). Proteins that remained bound to the column were eluted with drugs, and identified by mass spectrometry/bioinformatics. Lapatinib bound to Tb927.4.5180 (termed T. brucei lapatinib-binding protein kinase-1 (TbLBPK1)) while AEE788 bound Tb927.5.800 (TbLBPK2). When the NAD(+) wash was omitted from the protocol, AEE788, canertinib and lapatinib eluted TbLBPK1, TbLBPK2, and Tb927.3.1570 (TbLBPK3). In addition, both canertinib and lapatinib eluted Tb10.60.3140 (TbLBPK4), whereas only canertinib desorbed Tb10.61.1880 (TbCBPK1). Lapatinib binds to a unique conformation of protein kinases. To gain insight into the structural basis for lapatinib interaction with TbLBPKs, we constructed three-dimensional models of lapatinib•TbLBPK complexes, which confirmed that TbLBPKs can adopt lapatinib-compatible conformations. Further, lapatinib, AEE788, and canertinib were docked to TbLBPKs with favorable scores. Our studies (a) present novel targets of kinase-directed drugs in the trypanosome, and (b) offer the 4-anilinoquinazoline and pyrrolopyrimidines as scaffolds worthy of medicinal chemistry and structural biology campaigns to develop them into anti-trypanosome drugs.

Dataset Information

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

Publications

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

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