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With
current estimates placing the cost to develop a drug from
concept to the market at more than $800 million, pharma
companies are continually looking for new strategies to
gain more and better information about drug candidates,
their ability to bind a target, the results of those
interactions, and off-target effects as early as possible
in lead discovery and optimization. Technologies aimed at
extracting more and different types of data from
high-throughput screening (HTS) campaigns are reaping
rewards. Novel approaches to conducting biochemical,
functional, and ADME-tox assays in high-throughput formats
are generating lead compounds that come with a substantial
“resume” profiling their activity, specificity, and
pharmacokinetic (PK) and pharmacodynamic (PD)
characteristics.
“Industry
is making a conscious decision to perform cell-based
screening,” using functional assays to interrogate
target classes such as G Protein Coupled Receptors (GPCRs),
kinases, proteases, ion channels, and nuclear hormone
receptors, says Sailaja Kuchibhatla, senior vice president
of business development at DiscoveRx Corp. Technology can
aid in this process, she says, “by allowing screeners to
use current instrumentation, maintain throughput, and
allow for minimal perturbation to cells,” as well as to
choose from a variety of cell types for assay
development.
DiscoveRx’s
PathHunter cell-based assay platform performs
luminescence- or fluorescence-based homogeneous assays in
a microtiter plate (96, 384, or 1536) format using
conventional plate readers. It supports a range of
functional assay types, including translocation,
degradation, secretion protein, protein interaction, and
membrane trafficking.
The
basis for PathHunter is DiscoveRx’s Enzyme Fragment
Complementation (EFC) technology, in which beta-galactosidase
is divided into two parts: a larger inactive component of
the enzyme called the Enzyme Acceptor (EA), and a smaller
peptide called Prolabel. When separate, the enzyme is
inactive, but when brought together in the same cell
compartment—as a result of translocation of a target
protein when activated by its ligand—the two components
undergo high-affinity complementation to reconstitute a
functional beta-gal enzyme, leading to substrate turnover.
The
company recently expanded its family of PathHunter ß-Arresting
chemiluminescence GPCR assays that measure activation of
Gi, Gs, or Gq-coupled receptor signaling via direct
arrestin recruitment. The technology provides “a
universal way to look at GPCR signaling,” says
Kuchibhatla, as arrestin is recruited when any GPCR is
activated.
Kuchibhatla
projects growing interest in the future in profiling
services that rely on cell-based assays to test a set of
compounds against a variety of cell types, for example to
predict off-target effects.
High Content Meets High
Throughput
At a
recent cell-imaging conference, Evotec Technologies and F.
Hoffmann-La Roche presented a joint poster describing the
combined application of the Evotec Opera confocal laser
plate reader with a plate::explorer UHTS system for
high-throughput, high-content screening (HCS).
The
industry is looking for “smaller, more flexible
screening platforms” for HCS, says Andreas Niewöhner,
product manager for automated systems at Evotec. The
prototype instrument evaluated at Roche combines an HTS
workstation and image-analysis system. In April, Evotec
will launch a second-generation plate::explorer-like
instrument called the cell::explorer, which will also be
linked to the Opera reader and to automation hardware in
an integrated system designed for medium-throughput HCS.
This platform will offer capabilities appropriate for RNAi
screens, ADME-tox assays, and a range of primary and
secondary screening applications.
Screening
groups looking to adopt HCS want systems that are easy to
use and offer high throughput. TTP LabTech’s Acumen eX3
fluorescence multi-wavelength microplate cytometer can
analyze up to 300,000 data points in a 24-hour period in
1536-well format. The instrument provides three laser
excitation wavelengths—405, 488, and 633 nm—to expand
the range of fluorescent reagents that can be used for
multiplexed assays. It also employs whole-well scanning.
By scanning the entire well, the instrument can collect
data from each cell, rather than only a subset of cells,
helping to normalize variation within a well and across a
plate and facilitating rare event detection, such as stem
cell differentiation.
TTP has
demonstrated a range of applications for the Acumen eX3,
including cell cycle determination and quantum dot
analysis in combination with Qdot labeling to detect
multiple populations of antibody-bound monocytes in
parallel, for example. For GPCR screening using ß-lactamase
reporter gene analysis, the instrument requires reduced
cell numbers compared with those needed to perform bulk
fluorescence assays. Using an assay to measure dopamine
D1-receptor activity, for example, the instrument is able
simultaneously to measure dopamine D1-receptor activity
and cell number in each well, enabling correlation of
compound activity with cytotoxicity.
As
recently as ten years ago, researchers were not able to
use assay endpoints such as translocation or cell
morphology, observes Wayne Bowen, chief scientific officer
at TTP LabTech. “We do a lot of work in protein kinase
screening,” for example, Bowen says. Some protein kinase
targets are not amenable to binding assays and require the
ability to detect translocation.
Bowen
points to the combination of new instruments and reagents
as driving advances in HCS. For example, in neuroscience,
a novel fluorescent reagent is allowing researchers to
study serotonin reuptake inhibitors by tracking serotonin
uptake. And in oncology, where the goal is to kill cancer
cells, HCS enables cell cycle determination and cell
proliferation assays.
With
the ability to probe ligand/target binding events down to
the single-cell level, SRU Biosystems’ BIND technology
is a label-free, biosensor-based quantitative method for
directly measuring binding of small molecules to
immobilized protein targets in a microplate format.
Owen
Dempsey, CEO of SRU, describes the approach as a
combination of miniaturization, nanotechnology, and
advanced optoelectronics
technology applied to the detection of a biological event
without the need to tag either the small molecules or the
protein targets. An optical biosensor embedded in the
bottom of microplates produces a baseline wavelength that
shifts when small-molecule compounds are added to the
wells and binding occurs. The BIND Reader can process 96-
and 384-well plates, and the BIND Scanner, a 1536-well,
high-resolution, image-based instrument, is in
development.
“Companies
are looking for new technologies that can help screen
against novel target classes such as GPCRs and kinases,”
says Dempsey. They also want more information about the
biology, mechanism of action, and stoichiometry of binding
events and to be able to do pathway profiling.
Predicting selectivity
and toxicity
Ambit
Biosciences’ most recent drug discovery and development
collaboration, with Cephalon, focuses on kinase inhibitors
and leverages Ambit’s KinomeScan technology for
screening chemical libraries across a panel of more than
300 kinases. Highly selective kinase inhibitors, such as
the Novartis drug Gleevec, used to treat patients with
chronic myeloid leukemia (CML with a bcr-abl
translocation), can be highly effective and safe. But many
compounds will cross-react with multiple members of this
large family of bioactive enzymes, and much remains to be
learned about the safety, efficacy, and selectivity of
small-molecule kinase inhibitors.
For
now, at least, there are no reliable methods for
predicting the selectivity of compounds—“it has to be
done experimentally,” says Patrick Zarrinkar, senior
director of technology development and alliance management
at Ambit. The company screens compounds against a large
panel of kinases—currently, 317 kinase assays—and uses
the information to annotate chemical libraries. Human
kinases are fused to a T7 bacteriophage and a known ligand
is immobilized on the solid support. Kinase/ligand binding
is then measured in the presence and absence of test
compounds.
“We
see a wide range of selectivity,” says Zarrinkar.
“Some compounds are quite selective and some quite
promiscuous. You can be misled if you only screen against
a select number of kinases. You need to understand the
relationship between chemical structure and different
classes and subclasses of kinases.”
Ambit
employs an ATP-site dependent competition binding assay
using a known ligand. Zarrinkar anticipates that future
advances in the assay technology will include the
exploration of allosteric kinase inhibitors that interact
outside the ATP binding site, and the development of
assays for lipid kinases.
In
vitro screening
strategies designed to assess potential toxicity of new
drug candidates can help reduce the risk of late-stage
attrition. The key to successful in
vitro testing is to develop models with in
vivo correlates, so the in-vitro
data has relevance to subsequent animal studies.
CeeTox
uses a cell-based systems biology approach to evaluate
toxicity. The company developed its toxicity profiling
algorithm “to synthesize a large amount of in
vitro data (in cells) to provide an estimated blood
concentration where toxicity would be expected to occur in
rodent studies,” explains James McKim, Ph.D., president
and CEO. During the hit-to-lead and lead-optimization
stages, in vitro
toxicity screening allows medicinal chemists to monitor a
drug’s intended properties along with toxicity as they
modify a compound’s structural properties. By
identifying biochemical risk profiles of drugs that make
it to the market and are either withdrawn or limited in
their application due to unanticipated side effects in
defined subpopulations, researchers can begin to correlate
“more subtle changes with more chronic and low incidence
toxicity in the patient population,” McKim adds.
In
vitro tox
screening and in silico modeling are “already
reducing animal testing,” says McKim, and their
predictive power continues to improve.
The
CeeTox algorithm incorporates multiple-endpoint analysis,
using nine biochemical assays to assess dose-response
profiles, Pgp interaction, solubility, metabolic
stability, and in
vivo validation to produce an estimate of the
sustained blood concentration in a rat 14-day repeat dose
study at which toxicity would first be expected to occur.
The result of HTS is an estimate of in
vivo toxicity, information on the mechanism of
toxicity, and the ability to rank order hits based on both
potency and toxicity. Additional predictive information
can be developed by incorporating assays that evaluate
cytochrome P450 (CYP) enzyme induction, metabolic
activation, cardiotoxicity, endocrine interactions, and
species-specific metabolism.
Copyright 2007, Cambridge Healthtech
Institute. All Rights Reserved. |
