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Leveraging the Promise of Chemical Genomics
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Stuart Schreiber of Harvard University and the Broad Institute. Source: Len Rubenstein
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inferring the effect on proteins, which
are the typical interaction partners for
small molecules. But that correlation is
imperfect. “The main shortcoming today
is that we don’t have a way to test therapeutic hypotheses in physiologically
relevant conditions with small molecules,”
Schreiber explains.
One challenge here is developing small
molecule libraries that represent biological
diversity. The chemical space of small
molecules is nearly infinite, exceeding
1060, more molecules than could be synthesized or screened. Still, many biologically
relevant classes of molecules are not wellrepresented in current libraries, especially
complex natural products (See sidebar:
Library bias and enhancing diversity).
As a result, research efforts continue in
the attempt to fill the gaps in biologically
relevant chemical space. Schreiber’s group
at the Broad Institute, as well as other
groups, are now using one approach called
diversity oriented synthesis to construct
new libraries, with the NIH supporting
a variety of this research through their
chemical methodology and libraries development (CMLD) initiative.
Part of the idea is to tap into the
creativity and resources of synthetic organic
chemists, who previously might not have
been involved in biology, says Jeffrey Aubé
of the University of Kansas, a principal
investigator on one of the CMLD grants.
Working initially with several PIs whose
groups develop new synthetic methods and
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High-throughput screening (HTS)
was once the exclusive domain of large
pharmaceutical companies. But with the
NIH’s Roadmap initiative, the Molecular
Libraries Program (MLP), alongside
publicly available libraries, today academic
and government laboratories have become
centers for looking at small molecules
as probes of biological function, and are
even taking steps toward developing lead
molecules for drug discovery. In many
ways this shift makes sense, this herculean
effort feeds on an open cross collaboration
among chemists, biologists, and informatics
experts.
Initial work that married synthetic
chemistry and biological assays with HTS is
beginning to pay dividends, both in innovations that allow researchers to directly test
the therapeutic benefit of molecules in
physiologically relevant systems and in the
development of small molecules that tweak
activity within individual cells. “I think it’s
stunning. The progress of the MLP during
this short period of time is remarkable,” says
Stuart Schreiber of Harvard University and
the Broad Institute.
Although these efforts are showing
promise, real challenges lie ahead. With the
vastness of chemical space, researchers are
trying to increase the diversity of chemical
structures available for screening. And
as more HTS datasets emerge, further
chemistry is needed verify and optimize
those hits to produce biological probes or
lead compounds for drug discovery.
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NIH’s Molecular Libraries Program is
giving chemical genomics researchers the
tools to develop molecules to probe cellular
function. But will all pieces of this herculean
effort be enough to bridge the chasm toward
translational research?
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Mining chemical diversity
According to Schreiber, most compounds
fail in the clinic not because they didn’t
engage and modulate a target of interest,
but because that process either didn’t lead to
efficacy or it produced unexpected toxicity.
“We’re really bad at making predictions,”
he acknowledges.
Many of those early predictions came
from looking at nucleic acid targets and
Vol. 52 | No. 1 | 2012
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synthesize compounds with novel structures, Aubé and his colleagues have the
resources to further advance these efforts
into compound libraries that can be used for
high throughput screening. After verifying
that the molecules and structure represent
nominally new chemical space, a postdoc
or graduate student from the synthetic
group might work with his staff to produce
a library of 120-140 compounds which are
prepared under quality controlled conditions for screening applications. The information is deposited in the public database,
the Molecular Libraries Small Molecule
Repository (MLSMR).
Screening at multiple
concentrations
While building library diversity is useful,
researchers are also designing screens to
elicit maximum information, and using
cheminformatics to analyze and make
predictions from the resulting data.
Early drug discovery screens typically
tested small molecules at a single cutoff
concentration as a way to screen a maximum
number of compounds quickly and
inexpensively. James Inglese of the NIH’s
Chemical Genomics Center questioned
that approach.
“My thought was, well, why are we
screening at just one concentration?” he
says. “You get a lot of false positives, or
you miss things.” Instead, Inglese and his
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colleagues developed a new method, called
quantitative high throughput screening
(qHTS), which can screen across up to
five orders of magnitude difference in the
chemical concentrations. The result is a
dataset of titration curves which allows
researchers to begin to see pharmacological
trends such as the IC50 and EC50 within
their initial screens.
In one recent application of qHTS,
Inglese and his colleagues married qHTS
a biological assay of the malaria parasite,
Plasmodium falciparum, developed
by Inglese’s colleagues at the National
Institute of Allergy and Infectious Disease
(NIAID) that can be used with their low
volume 1536-well microtiter plates. By
using this strategy to examine the effect
of library compounds on parasite viability,
and because of the genetic variability of
parasites from different locations, the data
has helped the researchers pinpoint the
basis of some of these response differences
and look at resistance factors to particular
drugs (1,2).
Chemistry to follow up
on the screen
Chemistry is also important after the
screen. An initial high throughput screen
produces a number of hits, molecules that
act on a target in some way. But this is just
the beginning; these molecules need to be
validated and optimized, and that’s where
medicinal chemistry centers play a role in
the Molecular Libraries Probe Production
Centers Network (MLPCN): Aubé
directs the Kansas Specialized Chemistry
Center and Vanderbilt University has its
Specialized Chemistry Center for Accelerated Probe Development
Once that initial screen is complete,
Aubé and his colleagues join forces with
a team composed of researchers from the
screening centers and assay developers to
decide which initial hits should be pursued
using confirmatory assays. It is then that
the medicinal chemists will follow up on
those hits and construct structure-activity relationships. “There are milestones
and targets of potency and selectivity and
physical properties that are associated with
each of the probes,” Aubé says.
Probe molecules can also feed back into
investigations of the molecular mechanisms that generated the hits in the first
place. Many of the screens are phenotypic:
researchers are looking for a desired cellular
response and identify hit molecules that
generate that response. But most researchers
would also like to go beyond phenotype to
better understand the biochemical pathways
that generated a particular response.
Vol. 52 | No. 1 | 2012
After all this work is complete, the
results are published as probe reports on
the MLP website and purified samples
are kept available for other researchers to
use. One probe report published by the
group last year details a molecule with a
novel structure that blocks the activity of
CDC42, a GTPase that regulates the cell
cycle. Since late February, Aubé estimates
he’s received a dozen requests for this
particular probe.
“As the network has gone forward, we’ve
been making a deliberate effort to ensure
that we’re providing not only compounds
that are going to inform basic biology, but,
when it’s reasonable to do so, also provide
compounds that might be the first step of a
lead compound for drug discovery or translational research.” The follow-up gap
Outside the specialized groups that
NIH has funded to carry out medicinal
chemistry work, finding the resources to
follow up on initial screening hits can
present a bottleneck in chemical genomics
research, says Hakim Djaballah, Director
of the High Throughput Drug Screening
Facility at Memorial Sloan-Kettering
Cancer Center. Even with these NIH
funded centers, the resources devoted to
this piece of the puzzle are relatively small
compared with the number of screening
centers that are producing hits from high
throughput screening. Following up on
screening hits is risky and is not necessarily hypothesis driven, which can make
it difficult to secure funding. “I’m involved
in at least 3 or 4 projects like that that are
tangibly parked because we don’t have any
money to move them forward,” Djaballah
says.
Even once medicinal chemistry is
done there’s still the question of doing
secondary and tertiary screening on the
optimized compounds. “The professor
who develops the assays then has to come
up with the funding for the secondary and
tertiary assays and the functional assays
to discern whether these [optimized]
compounds are biologically relevant,” notes
Rathnam Chaguturu, director of the High
Throughput Screening Laboratory at the
University of Kansas.
Non-profit foundations with an interest
in a particular rare disease can form one
potential source of funding for projects
aimed at questions in particular disease
areas. Inglese, who studying Charcot-MarieTooth disease, a rare peripheral neuropathy,
has funding from a foundation that supports
this research. Another approach that could
shorten the process of lead optimization is to
start with a smaller pharmaceutical library
of known drug compounds, such as the one
NCGC assembled and published in Science
Translational Medicine (3). That process of
repurposing known drugs, whose potency
and activity have already been thoroughly
Library bias and enhancing diversity
In the early days of high-throughput screening (HTS), a lot of potential hits turned
out to be artifacts. Brian Shoichet and his colleagues at UCSF saw similar patterns
in their own structure-based screening work and started to wonder if the problem
was with the methods being employed or in the molecules that were being used for
screens.
Around the same time, Shoichet became aware of articles estimating the vastness
of chemical space, which led him to another question: with the limited number
of compounds available, why does high throughput screening ever work at all? In
2009, Shoichet and his colleagues published a computational analysis revealing that
existing libraries are biased toward biogenic molecules (4). Molecules in existing
libraries tend to be particularly good for targeting G-protein coupled receptors, ion
channels, and kinases. Therefore, if researchers blindly increase chemical diversity,
the likelihood of hits might actually get worse. A better strategy is to think in terms
of the molecules and scaffolds organisms likely have in their environments, such as
natural products.
The challenge with natural products is that many have not been synthesized,
and it turns out they are often difficult to isolate in sufficient quantities from the
organisms that produce them. One emerging solution is to use natural products
extracts, mixtures of compounds that are isolated from microorganisms (5). But
producing extracts that are clean enough and homogenous enough for screening
is complicated. Such extracts might include thousands of compounds of similar
molecular weight, yet only a handful might be active in a screen. According to Brian
Bachmann of Vanderbilt University isolation is the real problem. “That’s where we’re
putting our energy right now. If you had a method that could beam compounds out
of an extract and put them in tubes, that’s what we need.” -SW
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Quantitative high throughput screening (qHTS) allows researchers to obtain dose-response relationships during their initial screen
of compounds. By varying the concentrations of compounds being screened, researchers can quickly construct response curves.
Based on these patterns of activity, they can group compound classes based on their activity profiles and build structure-activity
relationships.
Vol. 52 | No. 1 | 2012
1. Yuan et al. 2009. Genetic mapping of targets
mediating differential chemical phenotypes
in Plasmodium falciparum. Nature Chemical
Biology 5, 765.
2. Yuan et al. 2011. Chemical genomic profiling
for antimalarial therapies, response signatures,
and molecular targets. Science 333, 724.
3. Huang et al. 2011 The NCGC Pharmaceutical
Collection: A comprehensive resource of clinically approved drugs enabling repurposing and
chemical genomics. Sci Trans Med. 3, 1.
4. Hert et al. 2009 Quantifying biogenic bias in
screening libraries. Nature Chemical Biology
5, 479.
5. Cruz et al. 2011. Titration-based screening for
evaluation of natural product extracts: Identification of an aspulvinone family of luciferase
inhibitors. Chemistry & Biology 18, 1442.
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tested, for new indications provides a fast
track to the clinic. “A disease foundation
wants that,” says Inglese. “They could use
that information to begin a clinical trial
with patients.”
For those molecules that might have
clinical applications, researchers and institutions would like to hold on to the intellectual property rights, but use of the NIH
Molecular Libraries Program resources
requires researchers to deposit the structures of the molecules that they discover
into a public database. “By definition
and by mandate, once done you have to
post the structures into Pubchem. And
once you’ve posted in Pubchem database,
you’ve lost your intellectual property,”
notes Chaguturu. “That’s one of the biggest
drawbacks.”
Although currently funded through
2014, questions surrounding future funding
for the Molecular Libraries Program loom.
High throughput screening is expensive,
and it’s a kind of business transaction in the
academic world, says Djaballah. “The initial
investment is written off by a university, but
maintaining [HTS centers] is expensive.”
“This network has put into place some
really fantastic infrastructure and has
enabled some tremendously talented scientists from all over the country to engage
in this work,” says Aubé. “It would be
wonderful to see those capabilities leveraged
in the future, and I’m not exactly sure how
that will happen.”
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