Abstract
Activation of the heat shock response, and in particular upregulation of stress-inducible Hsp70, herein referred to as Hsp70i, in newly transformed cells, appears to protect against protein damaging stimuli, induction of premature oncogene-induced terminal senescence (OIS), and apoptosis, thereby enabling tumor initiation and progression to an aggressive phenotype. Expressed at very low or undetectable levels in normal tissue, the cytoprotective effects of Hsp70i appear to be mediated through its activity as a molecular chaperone allowing proper folding of mutated proteins, and by blocking cell signaling pathways that regulate OIS and apoptosis. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. However, identification of selective inhibitors of Hsp70i has proven challenging largely because of the affinity of the protein for ATP. Additionally, its chaperone functions do not lend the protein amenable to traditional enzymatic high-throughput screens. Here, we describe the use of fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify Hsp70i inhibitors. The FLECS assay is a simple binding assay that enables proteins tagged with fluorophors to be rapidly and quantitative screened against small-molecule libraries. We show several case history examples of the methodology that led to the discovery of the Fatty acid synthase inhibitor, FASNALL, the DAPK3 inhibitor HS38, and HS72, an allosteric inhibitor selective for Hsp70i.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Daugaard M, Rohde M, Jaattela M (2007) The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions. FEBS Lett 581:3702–3710
Evans CG, Chang L, Gestwicki JE (2010) Heat shock protein 70 (hsp70) as an emerging drug target. J Med Chem 53:4585–4602
Hunt C, Morimoto RI (1985) Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70. Proc Natl Acad Sci U S A 82:6455–6459
Dix DJ, Allen JW, Collins BW et al (1996) Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. Proc Natl Acad Sci U S A 93:3264–3268
Wacker JL, Huang SY, Steele AD et al (2009) Loss of Hsp70 exacerbates pathogenesis but not levels of fibrillar aggregates in a mouse model of Huntington's disease. J Neurosci 29:9104–9114
Tavaria M, Gabriele T, Kola I, Anderson RL (1996) A hitchhiker’s guide to the human Hsp70 family. Cell Stress Chaperones 1:23–28
Ramos C (2011) Molecular chaperones and protein quality control. Protein Pept Lett 18:100
Mayer MP, Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62:670–684
Powers MV, Jones K, Barillari C, Westwood I, van Montfort RL, Workman P (2010) Targeting HSP70: the second potentially druggable heat shock protein and molecular chaperone? Cell Cycle 9:1542–1550
Swain JF, Dinler G, Sivendran R, Montgomery DL, Stotz M, Gierasch LM (2007) Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker. Mol Cell 26:27–39
Powers MV, Clarke PA, Workman P (2009) Death by chaperone: HSP90, HSP70 or both? Cell Cycle 8:518–526
Massey AJ (2010) ATPases as drug targets: insights from heat shock proteins 70 and 90. J Med Chem 53:7280–7286
Qi R, Sarbeng EB, Liu Q et al (2013) Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP. Nat Struct Mol Biol 20(7):900
Fewell SW, Day BW, Brodsky JL (2001) Identification of an inhibitor of hsc70-mediated protein translocation and ATP hydrolysis. J Biol Chem 276:910–914
Braunstein MJ, Scott SS, Scott CM et al (2011) Antimyeloma effects of the heat shock protein 70 molecular chaperone inhibitor MAL3-101. J Oncol 2011:232037
Propper DJ, Braybrooke JP, Taylor DJ et al (1999) Phase I trial of the selective mitochondrial toxin MKT077 in chemo-resistant solid tumours. Ann Oncol 10:923–927
Britten CD, Rowinsky EK, Baker SD et al (2000) A phase I and pharmacokinetic study of the mitochondrial-specific rhodacyanine dye analog MKT 077. Clin Cancer Res 6:42–49
Leu JI, Pimkina J, Frank A, Murphy ME, George DL (2009) A small molecule inhibitor of inducible heat shock protein 70. Mol Cell 36:15–27
Taguwa S, Frydman J (2015) The significance of Hsp70 subnetwork for Dengue virus lifecycle. Uirusu 65:179–186
Howe MK, Bodoor K, Carlson DA et al (2014) Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70. Chem Biol 21:1648–1659
Fadden P, Huang KH, Veal JM et al (2010) Application of chemoproteomics to drug discovery: identification of a clinical candidate targeting hsp90. Chem Biol 17:686–694
Haystead TA (2006) The purinome, a complex mix of drug and toxicity targets. Curr Top Med Chem 6:1117–1127
Graves PR, Kwiek JJ, Fadden P et al (2002) Discovery of novel targets of quinoline drugs in the human purine binding proteome. Mol Pharmacol 62:1364–1372
Alwarawrah Y, Hughes P, Loiselle D et al (2016) Fasnall, a selective FASN inhibitor, shows potent anti-tumor activity in the MMTV-Neu model of HER2(+) breast cancer. Cell Chem Biol 23:678–688
Carlson DA, Franke AS, Weitzel DH et al (2013) Fluorescence linked enzyme chemoproteomic strategy for discovery of a potent and selective DAPK1 and ZIPK inhibitor. ACS Chem Biol 8:2715–2723
Zeng XC, Bhasin S, Wu X, Lee JG, Maffi S, Nichols CJ, Lee KJ, Taylor JP, Greene LE, Eisenberg E (2004) Hsp70 dynamics in vivo: effect of heat shock and protein aggregation. J Cell Sci 117:4991–5000
Acknowledgements
This work was supported by NIH grants R01-AI089526-04 to T.A.J.H. and a Department of Defense Transformative Vision Award to T.A.J.H.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Haystead, T.A.J. (2018). Fluorescent-Linked Enzyme Chemoproteomic Strategy (FLECS) for Identifying HSP70 Inhibitors. In: Calderwood, S., Prince, T. (eds) Chaperones. Methods in Molecular Biology, vol 1709. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7477-1_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7477-1_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7476-4
Online ISBN: 978-1-4939-7477-1
eBook Packages: Springer Protocols