Introduction: What is Chemical Biology

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IntroductionWhat is Chemical Biology?
English presentation skills seminars in chemical
biology
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Chemical Biology is

• Chemical Biology seeks to develop new tools based
  on small molecules that allow minimal
  perturbation of biological systems while
  providing detailed information about their
  function.

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Why small moleculesif you can make knock-out
mice?

• Perturb protein function at given timepoint
• Temporal control
• No compensation effects
• Allows study of lethal knock-outs

• Lead compound for drug development

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Overview

• Small molecule screens looking for targets
• Proteomics identifying targets
• Probe design for enzymes smart targeting
• Imaging techniques visualizing targets

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Forward chemical genetics

• Libraries collection of small molecules

• Chemicals as mutagens

• Looking for phenotypes
• Easy readout
• High throughput (96-well, 384-well)

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Screening sifting

• Be aware of
• False positives

• False negatives

• Often several selectivity-filters
• 2nd or 3rd screen lower throughput

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Example
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Goal

• Get to know more about mitosis
• Only known small molecule inhibitors
• target tubulin

Taxol
Vinblastine
Green tubulin Blue chromatin
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Screening

• 1st screen cytoblot assay
• Detects post-translational modification
• in whole cells
• Low volumes (µl-nl)

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Cytoblot

• Culture cells in 384, 1536- or 6144-well plates
• Incubate with library compounds/controls

• Fix detect by antibody-HRP conjugate

Stockwell, Schreiber, Chem. Biol. 1999
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Screening for mitotic arrest

• Use antibody against Phospho-Nucleolin
• 139 of 16320 compounds show increase

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Second screen in vitro tubulin polymerization

• Why? We dont want tubulin-targeting molecules

• 86 show no effect

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Third screen fluorescent microscopy

• 86 compounds
• 27 no effect -gt false positives
• 42 effects in interphase mitosis
• 5 only affect mitosis

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One interesting compound Monastrol

• Result no spindle bipolarity

• Eg5 (a kinesin motor protein)
• involved in separation of
• centrosomes

• Not a general kinesin inhibitor
• organel localization normal

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Overview

• Small molecule screens looking for targets
• Proteomics identifying targets
• Probe design for enzymes smart targeting
• Imaging techniques visualizing targets

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Use small molecule as fishing hook

• Non-covalent binding to protein

• Make affinity-resin

Leslie, Hergenrother, Chem. Soc. Rev. 2008,
1347-60
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Enriching for your target

• Incubate with cell lysate/tissue homogenate
• Wash to eliminate non-binding proteins

• Elute
• Denature
• Add free ligand

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Use small molecule as fishing hook

• Covalent binding to protein

Affinity Matrix
Biotinylated molecule
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Streptavidin-mediated enrichment

• Streptavidin-Biotin
• KD 10-15 mol/L
• ? Efficient enrichment of
• diluted proteins

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Identify targets by Mass Spectrometry
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Tandem Mass Spectrometry

• Ionize fragment tryptic peptides

• Algorithm can assign
• peptide sequence search
• database

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Overview

• Small molecule screens looking for targets
• Proteomics identifying targets
• Probe design for enzymes smart targeting
• Imaging techniques visualizing targets

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Activity-based Proteomics

• Use enzyme mechanism gt covalent tagging
• Proteases nucleophile in active site

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Activity-based probes

• Covalent inhibitors with a tag

Cravatt et al., PNAS 1999, Bogyo et al., Chem
Biol. 2000
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Covalent modification of active site

• Only with active enzyme

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Profiling of protease activity

• Profiling of Protease Activity

Mouse Pancreatic Cancer Model Cathepsin
activities upregulated
Joyce et al., Cancer Cell 2004
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When its hard to get selectivity

• Kinases 491 kinase domains in human genome
• most drugs target ATP binding site

• 20 of kinases have small Thr- gatekeeper

Cohen, M. S, Taunton, J et al., Science 2005,
308, 1318
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Look for a second selectivity filter

• Cysteine in flexible loop in 11 kinases

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Inhibit specific kinases

• RSK2 has both Thr and Cys

Detection autophosphorylation of RSK2

• Mutating MSK1 gatekeeper sensitization

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Overview

• Small molecule screens looking for targets
• Proteomics identifying targets
• Probe design for enzymes smart targeting
• Imaging techniques visualizing targets

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Why imaging?

• Protein localization

• Detection of pathologies in vivo

• Most common GFP and derivatives
• Different colors available
• Large protein

• Alternatives
• Target small peptide sequences (modified protein)
• Target certain activity

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Targeting modified proteins
Protein Tag
Peptide Tag
Enzyme mediated Tag
Unnatural amino acid
Chen et al. 2005 Curr. Opin. Biotech. 16, 35-40
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Peptide tags

• Chemical fluorophores
• FlAsH
• (a ton more)

• FlAsH
• Tight binding
• Multiple colors

Tsien et al. 2000 Methods Enzymol.
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FlAsH application

• Study dynamic structures
• Gap junction plaques
• Small molecule signaling between cells
• Half life lt5 hours
• Tetracysteine tag on Connexin43

Tsien, Ellisman et al., Science 2002
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Enzyme mediated tags

• Enzymatically linking a fluorophore
• Trans-glutaminase
• Biotin ligase

Lin et al. 2006 JACS 128, 4542-4543
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Target certain activity

• Activity-based probe mediated imaging
• Clan CA proteases in Pancreatic cancer

Normal islets
Angiogenic islets
Tumors
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Is this all?

• Carbohydrate detection
• Detection of other post-translational
  modifications
• Probes to monitor Ca2 or other metals