Transcript Lecture 11, chemical genetics - Cal State LA

Uncovering the Function of a Gene:
Classical Genetics
In classical genetics, researchers generate mutations, then work
backwards to deduce the normal function of the mutated gene
Example:
- mutate many Drosophila fruit flies
- screen for mutants that live unusually long lives
- identify gene mutated in the long-lived flies (methusalah)
- study how the normal version of this gene shortens lifespan
Drawback: this approach is not practical for mammals like us
- random mutations hard to generate and pinpoint
- many redundant copies of key genes
- long generation times, ethical considerations limit experiments
Uncovering the Function of a Gene:
Chemical Genetics
In chemical genetics, researchers use small molecules to disrupt the
normal function of protein targets, then identify those targets
Example:
- the compound colchicine kills cells by blocking mitosis
- radioactively labeled colchicine bound to a protein in cells
that was later identified as tubulin
- this is how it was first discovered that microtubules are
polymers of a and b-tubulin
Advantages:
- small compounds can easily cross cell membranes
- can often be washed out to restore normal phenotypes
- can then serve as probes to isolate the target proteins
Chemical Genetics
Use natural products or synthetic molecules to induce a specific
phenotype in whole cells
-
- this approach has improved our understanding of:
- intra-cellular signaling pathways
- cell cycle progression
- proteins involved in specific disease states
Chemical Genetics
Thus, use small molecules as probes to link the genome (which is
information) to the proteome (which carries out actions)
- goal: understand the function of every protein
Having a specific inhibitor for every protein would give us great
control over what’s going on in a cell
- allow specific modulation of the proteins contributing to
a particular disease state, for instance
Identifying the Target for a Bioactive Molecule
Techniques for matching a small molecule to its target:
(1) Affinity chromatography
(2) Photo-affinity & chemical cross-linking
(3) Protein micro-arrays
(4) mRNA-protein fusions
(5) Drug Westerns
(6) Phage display libraries
Affinity Chromatography
Small molecule is derivatized, linked to a solid support
-
Column is loaded with derivatized solid support
- Incubated with solubilized proteins (target binds to column)
- Washed with a buffer to rinse off unbound proteins
Bound protein is eluted from column by washing with solution of
free ligand
-
Protein is then visualized by gel electrophoresis (coomassie blue or
silver staining)
Affinity Chromatography
Carbodiimide coupling is a
standard way to covalently
link molecules through
carboxyl and amine groups
Affinity Chromatography
How do you isolate
a dopamine binding
protein?
linked molecules
Photo-affinity & Chemical cross-linking
Instead of linking drug to a solid support, attach another molecule that
is reactive with light or protein functional groups (primary amines)
- This “linker” molecule will covalently bind the protein once the
drug binds (non-covalently) to its protein target
- Linker may be radioactive, so the protein gets labeled and can
later be visualized on a gel
Process:
- Drug + linker complex enters cell; drug binds to target
- Irradiated with light or allowed to spontaneously react

Photo-affinity & Chemical cross-linking
Photo-affinity & Chemical cross-linking
Step 1: react drug with linker, in a
test tube
known drug
*
unknown
protein
Photo-affinity & Chemical cross-linking
Step 2: add drug-linker to cell
- drug will bind (non-covalently) to its protein target
*
Photo-affinity & Chemical cross-linking
Step 3: shine light to activate photoreactive end of the linker,
which will covalently bond to the protein
UV light
photoreactive end
*
*
Photo-affinity & Chemical cross-linking
Step 3: shine light to activate photoreactive end of the linker,
which will covalently bond to the protein
- the photoreactive end also carries a radioactive label ( ),
which now marks the protein
*
*
unknown protein is now radioactive, will show up on film
as a spot after being run out on a protein gel
Protein Microarrays
Microarrays are tiny chips to which are attached a large number of
proteins
 proteins retain their enzymatic functions, and bind ligands
2 kinds of microarrays:
(1) protein function array
Each protein in a cell is expressed + attached to a defined spot on chip
- detects which attached protein(s) the added ligands bind to
- by adding a drug attached to a fluorescent marker, you can determine
what cellular protein(s) a labeled drug binds
Protein Microarrays
Microarrays are tiny chips to which are attached a large number of
proteins
 proteins retain their enzymatic functions, and bind ligands
2 kinds of microarrays:
(2) protein-detecting array
Chip is coated with diverse small-molecules, and washed with proteins
to see where binding occurs
- detects which attached drug a particular labeled protein binds to
protein function array
yellow dots:
different
bound
proteins
blue dots:
different
bound
drugs
extract A = red
label
extract B = green
label
protein-detecting array
Making Microarrays I
- plain glass slides derivatized to yield a sheet of maleimide groups
- maleimide reacts with any -SH group to form a covalent bond
- from combi-chem run, 1 bead placed in each well of a microtitre plate
Making Microarrays II
- compound from each bead released, individually spotted onto slide
by robot (200 mm spots, >1,000 spots per cm2 on slide)
- slide then probed with labeled fluorescent protein(s) to detect binding
MacBeath et al. 1999,
PNAS 121:7967
Trial run: 3 different compounds with known binding proteins
spotted onto a slide, in alternating fashion
- then probed with all 3 proteins, each with a different color label
- each spot was correctly bound and labeled by its cognate protein
Protein Microarrays: Detection
How do you detect to which spot on a chip proteins have bound?
(1) Tag proteins with Green Fluorescent Protein (GFP)
- this will cause all proteins to fluoresce under the right light
 incubate chip with solution of GFP-tagged cellular proteins
-
-
Protein Microarrays: Detection
How do you detect to which spot on a chip proteins have bound?
(2) Surface plasmon resonance
- no protein modification is necessary for detection
Uses a laser as a highly sensitive microbalance: detects tiny mass
differences from the backside of the chip, indicating which spots
have proteins bound to them
Can be used in tandem with mass spectrometry to detect binding events
and simultaneously determine the mass and the sequence of the bound
protein by MALDI-TOF MS and MS/MS -- in a single experiment!
- the future of protein microarrays
Protein Microarrays: Example
Kuruvilla et al. wanted to find a small molecule inhibitor of a known
protein, Ure2p (Nature 2002, 416: 653-657)
(1) Used diversity-oriented synthesis to make a library of 3,780 small
molecules
(2) Made a protein-detecting microarray: robotically spotted all
molecules onto a 4 cm2 glass slide
(3) Probed slide w/ fluorescently labeled Ure2p protein
- detected 8 spots, indicative of protein-binding
(4) 1 of 8 “hits” was found to intensely inhibit Ure2p protein; called
uretupamine
Protein Microarrays: Example
Kuruvilla et al. wanted to find a small molecule inhibitor of a known
protein, Ure2p
(5) Made of series of derivatives of uretupamine, found 1 w/ improved
inhibitory activity (uretupamine B)
(6) Used microarrays to probe the effects of inhibiting Ure2p on overall
gene expression
- discovered that only a subset of the genes controlled by Ure2p
protein are expressed when Ure2p is inhibited by this drug
- showed that small molecules can provide more information
about multi-purpose proteins than genetic deletions, by
selectively turning off some, but not all, protein functions
Protein Microarrays: Example
Process: Kuruvilla et al. used a series of methods we have discussed-(1) combinatorial chemistry
(2) protein-detecting microarrays
(3) pharmacophore-based optimization
-followed by(4) RNA-based microarrays + classical genetics, to explore effects of
Ure2p-inhibition on cellular physiology and gene expression
Identifying the Target for a Bioactive Molecule
Techniques for matching a small molecule to its target:
(1) Affinity chromatography
(2) Photo-affinity & chemical cross-linking
(3) Protein micro-arrays
(4) mRNA-protein fusions
(5) Drug Westerns
(6) Phage display libraries
Drawback:
Identifying the Target for a Bioactive Molecule
Techniques for matching a small molecule to its target:
(1) Affinity chromatography
(2) Photo-affinity & chemical cross-linking
(3) Protein micro-arrays
(4) mRNA-protein fusions
(5) Drug Westerns
(6) Phage display libraries
Advantage: these methods link
protein to its gene sequence
mRNA-Protein fusions
Technique for physically linking mRNA transcript to the end of each
protein
Attach the drug puromycin to 3’ end of all mRNA from a cell
Fusion proteins are made when ribosome reaches 3' end of mRNA
- Puromycin enters the peptidyl transferase site
- Creates a covalent link between the mRNA and new protein
Protein-mRNA fusions can then be screened for protein interactions
using affinity chromatography or other techniques
- the mRNA of bound proteins is reverse-transcribed and amplified
by PCR into a double-stranded DNA clone of the active protein
3’ end of mRNA is tagged
with the drug puromycin
Finished peptide ends up
covalently bound to end of
puramycin-mRNA fusion
Drug Western
Combination of 2 widely used cell biology methods:
1) western blots: proteins are attached to nitrocellulose filters,
screened with antibodies
2) library screening by colony lifts from plates of bacteria or phage
Protocol is akin to screening libraries with DNA probes, changed to
visualize protein-drug interactions
1. each colony is a bacterial clone containing a cDNA
insert; it will produce large amounts of its one protein
(and each colony likely has a different cDNA
insert, so will make a different protein)
2. blot onto a filter that will
trap the expressed proteins
1. each colony is a bacterial clone containing a cDNA
insert; it will produce large amounts of its one protein
(and each colony likely has a different cDNA
insert, so will make a different protein)
2. blot onto a filter that will
trap the expressed proteins
3. wash with your drug,
attached to something
visible (e.g., GFP)
4. go back to the plate and pick off the colonies
that produced binding proteins
Drug Western
Phage or bacterial cDNA library grown on agar plates, covered by
nitrocellulose filters
- each colony (spot on a plate) grew from a single cell carrying a
cDNA insert in a plasmid (different gene cloned into each colony)
- soak filter in isopropyl b-D-thiogalactopyranoside, which induces
expression of the inserted gene in each individual colony
Filters lifted from plates, washed and hybridized with a chemical probe
covalently attached to a marker molecule that visualizes binding
Once a positive plaque or colony is selected, the cDNA fragment
contained within is replicated, isolated and sequenced

Drug Western
Example: Identify binding protein of HMN-154
(anti-cancer drug w/ unknown
mechanism of action)
HMN-154 linked to protein BSA, used to screen
colonies
- antibodies to BSA used to probe for binding
Showed 2 proteins (NF-kB and thymosin b-10) were binding targets
of the drug
- confirmed by genetic knockout techniques
Tanaka et al. 1999
Phage Display Libraries
Create a library of cDNA sequences, with each cDNA inserted into a
different M13 bacteria virus (= phage)
- clone is positioned next to DNA encoding virus coat protein P6
Virus then transcribes a fusion protein linking the coat protein to the
unknown protein corresponding to the cDNA insert
- Fusion protein is placed on the surface of the viral capsid
(the protein shell encasing the viral genome)
In other words, the attached protein encoded by the cDNA clone is
presented on the outer surface of each phage particle
Phage Display Libraries
These virus particles are then used in affinity chromatography
- a drug has been linked to a solid support
- after chromatography, the “positive” phage (those that bind to
the immobilized drug on the column) are washed off
- these phage, which contain the clone of the binding protein, are
then amplified in bacteria
Constraints:
- protein must assume correct conformation on phage surface
- protein cannot inhibit virus from exiting a bacterial cell
- attachment of unknown protein to P6 cannot block binding site