title: Effects of cyclin-dependent kinase inhibitor Purvalanol B application on protein expression and developmental progression in intra-erythrocytic Plasmodium falciparum parasites.
abstract: BACKGROUND
  The 2013 Malaria World Report indicated that in 2012 there were approximately 207 million cases of malaria, which resulted in an estimated 627,000 malaria-related deaths. Due to the alarming resistance of these parasites to traditional anti-malarial treatments there is a pressing need to not only identify new anti-malarial compounds, but also to characterize the effect of compounds known to have an effect on the parasite life cycle. This study reports on effects of kinase inhibitor Purvalanol B administration on the growth and protein expression of Plasmodium falciparum late-stage trophozoites.
  
  
  METHODS
  A SYBR® Green I parasite growth assay was used to measure the IC50 of Purvalanol B with P. falciparum (strain W2). Purvalanol B or DMSO control were applied to synchronized parasites 36 hours post invasion and parasites were incubated for 12 hours. Giemsa-stained blood smears were used to determine the effect of Purvalanol B on parasite growth, global quantitative proteomic analysis was used to examine differences in protein expression between Purvalanol B-treated and control parasites and results were confirmed by qPCR.
  
  
  RESULTS
  There were no differences in parasitaemia between inhibitor-treated and control parasites. However, the ability of Purvalanol B-treated parasites to form schizonts was significantly reduced. Proteomic analysis detected 76 human proteins and 518 P. falciparum proteins (63 in control cultures only, 56 proteins in Purvalanol B cultures only, and 399 proteins in both cultures). Quantitative analysis of protein extracts revealed eight proteins that were up-regulated in the inhibitor-treated cultures, including several components of the parasite's proteasome complex and thioredoxin reductase. Two proteins appeared to be down-regulated, including a helicase and an RNA-binding protein.
  
  
  CONCLUSIONS
  Purvalanol B application decreases the ability of late-stage P. falciparum trophozoites to form multinucleated schizonts and up-regulates proteasome subunits and proteins that contribute to redox homeostasis, which may indicate an increase in oxidative stress as a result of inhibitor application. While the efficacy of Purvalanol B is relatively low for use as an anti-malarial therapy, quantitative proteomic analysis may serve as a method of examining the action of drugs on the parasite and indicate the likelihood of future resistance development.
authors: School of Biological Sciences, University of Northern Colorado, Greeley, CO, 80639, USA. kristen.bullard@gmail.com.
authors: Kristen M
authors: KM
authors: Bullard
authors: Proteomics and Metabolomics Facility, Colorado State University, Fort ColliHns, CO, 80523, USA. carolyn.broccardo@colostate.edu.
authors: Carolyn
authors: C
authors: Broccardo
authors: School of Biological Sciences, University of Northern Colorado, Greeley, CO, 80639, USA. susan.keenan@unco.edu.
authors: Susan M
authors: SM
authors: Keenan
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004912:Erythrocytes / Q000469:parasitology; D006801:Humans; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D020543:Proteome / Q000032:analysis / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics; D015800:Protozoan Proteins / Q000032:analysis / Q000145:classification / Q000235:genetics / Q000378:metabolism
chemical_list: D020543:Proteome; D015800:Protozoan Proteins; C113584:purvalanol B; D018844:Cyclin-Dependent Kinases; D000225:Adenine