Transcript The roles of chemical biology in drug development

Chemical biology –
the biologist’s perspective
Drug development 2007
Per Sunnerhagen
What are the goals of chemical biology?
• To develop chemical tools and applying them
to answer questions in cell biology
• The object is not to develop drugs, although results
obtained with chemical biology may well become
useful in drug development
The roles of chemical biology in drug development
Workflow of chemical biology at the Broad Institute, MIT
What arguments can be used for chemical, rather
than genetic, intervention as a tool for investigations
in cell biology?
1) Permanent shut-off (or overexpression) of a gene will cause secondary
adaptations in the mutant cell, leading to changes in e.g. expression
levels of other genes. By contrast, short-term inhibition using a chemical
will only produce primary effects
2) Certain cellular pathways and function (e.g. cytoskeleton, translation)
have a large proportion of essential genes, making genetic analysis difficult.
Chemical inhibitors open two possibilities for analysis of such pathways:
a) Partial continuous inhibition
b) Complete but transient inhibition,
in both cases preserving viability
3) Conditional mutants (e.g. temperature-sensitive alleles) require
conditions (e.g. temperature shift) that cause major changes in cell state,
making appropriate controls difficult to evaluate
cont’d
4) Continuous variability - by applying different amounts of inhibitor, it is
possible to get a graded response from an enzyme or a pathway.
This is essential for a systems biology analysis of a pathway, with the
intention of identifying the components, the activity of which are critical
for the overall output of the pathway.
Such components are potential drug targets.
5) Speed – it is possible to shut off the activity of a target (protein) within
seconds – a few minutes using small molecules as inhibitors.
Cf: transcriptional shut-off 30 – 60 minutes; RNAi hours – days.
Necessary to follow cellular events with fast kinetics – signal transduction,
cell cycle etc.
6) Selective inhibition of the interaction between a specific protein pair,
targeting one interaction domain, leaving other interactions intact.
This allows shutdown of only one branch of a pathway
7) Chemicals acting as fluorescent tags and e.g. ”caged substrates” can
be developed, adding further versatility
What about drawbacks?
• side effects (small molecules are never entirely specific; but
gene disruptions are)
• limited bioavailability: penetrating different cell types from different organisms
will vary; a small molecule developed will to some degree be speciesspecific
Protein kinases constitute a large protein family
The selectivity of kinase inhibitors can be evaluated using
panels of immobilised recombinant kinase enzyme
”Orthogonal chemical genetics” compared with ordinary genetics
Selective inhibition of
a genetically engineered
protein kinase allele using
a designed ”orthogonal”
inhibitor that can be
accommodated only by
the modified kinase.
All other ”wild-type”
kinases are unaffected
Analogue-sensitive kinase alleles can be used for fingerprinting the
response (e.g. transcript profile) to inhibition of a kinase. This can be
utilised in the search for new wild-type kinase inhibitor lead compounds
Analog sensitive kinase allele (ASKA)-based gene expression ‘blueprints’ for kinase inhibitor lead profiling.
Using the ASKA system, changes in gene expression resulting from potent, specific kinase inhibition can be
measured by DNA microarrays. The open circles represent cells with either ASKA or wildtype kinases; the
compounds are either the ASKA-specific orthogonal inhibitor or a lead compound of a wildtype kinase (lead).
The checkerboards represent gene expression patterns. The far-left treatment shows the pattern that results
from inhibition of the ASKA. The middle two treatments show the small changes that result from either the ASKA
or the analog inhibitor that can be ‘subtracted’ from the ASKA + inhibitor pattern. The far-right treatment shows
the pattern obtained by profiling a lead compound. By comparing gene expression patterns, such ASKA-based
blueprints can be used to determine the possible off-target effects of kinase inhibitor lead compounds.
In the example shown here, the lead compound gives the same profile as the ASKA blueprint; thus, the lead has
a similar potency and specificity to the ASKA.
Use of analogue-sensitive kinase alleles in mice
Analogue-sensitive kinase allele (ASKA) mice can be used either directly (in various chemically
induced disease models) or by mating to appropriate disease model strains. Specific kinase inhibition can
be achieved by injection of an orthogonal inhibitor that inhibits only the ASKA and not any wildtype
kinases.
Identification of protein kinase substrates using orthogonal mutations and
ATP analogues
Only the genetically modified
protein kinase – in this case
Src – will be able to accept the
bulky ATP analogues, which are
radiolabeled
As a result, only the direct
substrates of Src will be
radiolabeled, allowing their
identification
Second generation ATP analogues and orthogonal mutations
would be specific (excluding normal ATP from reaction)
Chemical Biology at Göteborg University
Current focus of the Chemical Biology Platform
Can we create several independent orthogonal pairs of
sensitive kinase alleles and inhibitors?
This would allow simultaneous and independent manipulations
in vivo of two or more protein kinases in a signalling pathway –
approaching true systems biology analysis
A chemical genetics approach to direct proteolysis to selected
targets in vivo (”chemical knockout”)
A heterofunctional synthetic molecule (PROTAC) includes ligand functionality for the target
protein (white triangle), a linker moiety (grey hourglass shape) and a ligand for E3 ligase
(black rectangle)
Expansion of the genetic code through incorporation of unnatural
amino acids through chemical genetics
Schematic representation of orthogonal chemical genetic strategy for site specifically incorporating unnatural amino
acids. An orthogonal synthetase is evolved that specifically acylates a cognate orthogonal suppressor tRNA with a desired
unnatural amino acid. To introduce an unnatural amino acid site specifically in a target protein, an appropriate codon is
mutated to an amber, opal or four base codon and transfected in the host cells. The orthogonal tRNA/synthetase pair in the
host cells responds to the mutated codon by incorporating the desired unnatural amino acid at the site of interest in vivo.
Biological uses of unnatural amino acids incorporated in
specific proteins
• a photo-crosslinking amino acid can be used to probe dynamic
protein-protein interactions at different stages of a signalling cascade;
transient enzyme-substrate complexes can be covalently bonded and
isolated
• fluorescent amino acids can be used to probe the microenvironment
created during the signalling processes (pH, redox etc.)
• amino acids bearing a photo-cleavable side chain provide a
temporal switch to regulate activity in vivo using laser
• ”killer” functionalities (e.g. azido groups) can be used to inactivate
a target enzyme at a specific time and place in the cell
• heavy atom-containing amino acids for protein structure studies
Incorporation of unnatural UV-crosslinkable amino acids into
protein can be used to reveal protein-protein interactions,
with high resolution in space and time
The bioorthogonal chemical strategy extended
A chemical reporter linked to a substrate is introduced into a target biomolecule through cellular
metabolism. In a second step, the reporter is covalently tagged with an exogenously delivered
probe. Both the chemical reporter and exogenous probe must avoid side reactions with nontarget
biomolecules.
Specific sequences of natural amino acids can work as a specific
base for chemical reporters
• Cys-Cys-X-X-Cys-Cys reacts selectively with biasenicals
This amino acid sequence can be introduced genetically into
any protein. This works in instances where GFP tagging disrupts
function of the protein.
HeLa cells expressing tetracysteine-fused connexin
were treated with FlAsH (green), incubated for 4
hours, then treated with ReAsH (red).
This two-color pulse-chase labeling experiment
shows that newly synthesized connexin is
incorporated at the outer edges of existing gap
junctions
Fluorescent probes: monitoring enzyme-substrate intermediates by
FRET (fluorescence resonance energy transfer)
(Top)
A photolabile chemical protection group
("cage") on the phosphate moiety of the
phosphotyrosine-containing synthetic
peptide prevents binding to the active
site of the PTP.
(Middle) UV-induced photolysis of the
cage induces substrate binding at the
PTP active site and FRET, monitored
by FLIM and/or emission intensity
changes.
(Bottom) After catalysis, the reaction
product dissociates from the PTP,
resulting in loss of FRET.
Yudushkin et al., Science 315:115 (2007)
“A map of Km/S for the enzyme (PTP1B)
reveals that the peripheral pool, near the
plasma membrane, of PTP1B
operates in a near-saturation regime
(i.e., low Km/S)”
Yudushkin et al., Science 315:115 (2007)
Combining genetics and chemical screening
A compound that causes cell death on its own can be used in a
genetic suppressor screen that identifies cDNAs (or siRNAs) that
prevent death caused by the compound. Such suppressor reagents
are likely to encode proteins involved in the response to the compound,
including the direct protein target of the compound
Mutants
Classification of crude extracts from
nature containing bioactive compounds
Clustering of mutants and chemicals
according to sensitivity profiles groups compounds
with similar mechanisms of action
Cell wall (staurosporin inhibits PKC;
caspofungin inhibits synthesis of -glucan)
Chemicals
Crude extract clusters together with
pure chemicals
Synthesis of ergosterol (membrane lipid)
DNA-damaging compounds
(Data are cleaned through eliminating mutants in membrane
pumps and membrane lipid metabolism, which give
multidrug sensitivity)
Parsons et al., Cell 126:611 (2006)
Factoring analysis finds correlations between compounds
based on responses in only a small cluster of genes
Verrucarin
and neomycin
both inhibit
translation.
They are
grouped
together in a
factoring
analysis but
not with
hierarcical
clustering.
Mutants in
this group
affect the
40S subunit of
ribosomes
Actinomycin, a
DNA-damaging
compound, which also
inhibits RNA
polymerase,
is grouped
here together with
other DNA-damaging
compounds
High correlation
between sensitivity
profiles for
extracts 00-192
and 00-132
Active compound
of extract 00-192
Active compound
of extract 00-132
Spontaneously
mutated strains
co-resistant to
both chemicals