13-Peter Karuso.pptx - C-HPP

Small Molecule Approaches for Protein Discovery
Peter Karuso
Inaugural C-HPP Human Proteome Missing
Proteins Workshop, July 30, 2014
Department of Chemistry & Biomolecular Sciences
Piggott, A.M. and Karuso, P. (2004), Comb. Chem. High Throughput Screen., 7, 607-630.
Anatomy of a chemical probe
Piggott and Karuso, Mar. Drugs (2005), 3, 36‐63. Activity‐based probes; Fluorophosphonates
Cravatt and co-workers, PNAS, 2002, 99, 10335.
Profiling serine proteases in cancer cell lines
Affinity‐based selection; Didemnin B
Affinity vs Concentration
EF‐1
EF‐1
PPT
palmitoyl protein thioesterase
Schreiber and co‐workers JBC
(1994) 269, 15411‐4
PNAS (1996) 93, 4316‐4319 Biotin pull‐down method
O
O
CH3
CH3
OCH3
O
OCH3 O
H
O
N
O
O
OH
O
O
(1)
O
O
(2)
OCH3
Cl
O
CH3
O
O
O
O
N
H
HN
H
H
N
2
NH
H
S
O
(3)
(1) Fumagillin (potent inhibitor of
angiogenesis)
(2) TNP-470
(3) Biotinylated probe
MetAP-2
Ny Sin, Lihao Meng, Margaret Q. W. Wang, James J. Wen, William G. Bornmann and Craig M. Crews. “The anti‐
angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP‐2” Proc. Natl. Acad. Sci. USA (1997) 94, 6099–103
Modern Chemical Proteomics Probes
Linker Architecture
“Smart Probes”
• specificity • coverage
• sensitivity
Capture Motif: recognition epitope; activity covalent label; cross‐linker Analysis Handle: affinity anchor; fluorescence tag; isotope labels
Regulated release: physical; chemical; biochemical
Modern Chemical Proteomics Probes
trifunctional probes from Liu lab
New multi‐functional probes
fluorescent tracer
Cross‐linker
*
photoactivated
release or binding
affinity anchor
*
13C isotope labels
spacer
protein binder
Modern Chemical Proteomics Existing nonspecific probes
Probes
targeting motif Dolai, S.; Xu, Q.; Liu, F.; Molloy, M. P. Proteomics
(2011), 11(13), 2683‐2692
More specific targeting motifs
O
OH
F
O
O
NH
HN
OH
O
OH
OH
OH
F
O
CO2H
O
NH
HN
OH
O
O
OH
Patel, A. R; Hunter, L.; Bhadbhade, M. M.; Liu, F. European Journal of Organic Chemistry, 2014(12), 2584‐2593
OH
CO2H
Karuso, P., “Modern Methods for the Isolation of Natural Product Receptors” Comprehensive Natural Products Chemistry II (Mander, L., Lui, H.‐W., Eds), Elsevier, Oxford, 2010, Vol. 9, pp513–567 Reverse Chemical Proteomics: Phage Display
• Since the cDNA insert is after the coat
protein gene, the STOP codon problem
is eliminated
T7 gene cp10
EcoR I
stop
codon
cDNA Insert T7 gene
Hind III
Reverse Chemical Proteomics: Phage Display
Red = strepavidin
Yellow = biotin
Reverse Chemical Proteomics: Yeast Display
Hemagglutinin
Aga2p
biotin
streptavidin
S S
S S
Aga1p
Yeast cell wall
Enrich binding clones through several rounds of FACS
Plasmid recovery from yeast cells, DNA sequencing, database search Reverse Chemical Proteomics Probes
Reverse Chemical Proteomics Probes; artesunate
BAD
Cell/Tissue Profiling using fluorescence activation suicide substrates
R = PO3H2 – phosphatase
SO3H – sulfatase
Glu – glycosidase
GlcNAc – specific glycosidases
COPr – esterase
“Enzyme tissue imaging”?
Active site lysine mapping
Cell/Tissue Profiling using Affinity Reagents
• Staurosporin binds to 95% of all known kinases in ATP binding pocket of kinases (3 overlays) • linker can be attached to the amino sugar (arrow)
• This would make a kinase profiling reagent
• The phthalimido derivative is fluorescent
Missing olfactory receptors
Linda Buck, Richard Axel Nobel Prize in Physiology and Medicine in 2004.
Missing olfactory receptors
Rasmussen, Nature (2011), 469(7329), 175‐
180.
β2AR receptor
Thompson, A. A. et al. Nature (2012), 485, 395–399. Wu, H. et al. Nature (2012), 485, 327–332.
Manglik, A. et al. Nature (2012), 485, 321–326.
Granier, S. et al. Nature (2012), 485, 400–404.
-opiate
receptor
Missing olfactory receptors
Volatiles responsible for the passion fruit notes of NZ Sauvignon Blanc
Gelis L, Wolf S, Hatt H, Neuhaus EM, Gerwert K. Prediction of a Ligand‐binding Niche within a Human Olfactory Receptor by Combining Site‐directed Mutagenesis with Dynamic Homology Modeling. Angew Chem Int Ed Engl. 2012, 51(5), 1079.
Significance
• targeting low‐abundance proteins by chemical proteomics increases the coverage and accuracy of the proteome
• Small molecule activity probes offer different profiles of cells and tissues to complement other ways of profiling
• Reverse chemical proteomics are fast and iterative, allowing amplification and selection of the most avid binders
• Reverse chemical proteomics does not require mass spectrometry – analysis is done at the gene level