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 Guest Commentary: Prioritizing hits based on drug-target residence time
September 2010
SHARING OPTIONS:
The assays used involve a careful balance between simplicity
(which inversely correlates with time and expense) and biological relevancy,
and should ensure (or at least not contradict) a correlation between assay
response and ultimate in vivo drug
action. While it is easy to build and perform robust assays to measure the
action of a soluble, unmetabolized compound on a single, well-characterized,
recombinantly expressed protein target, doing so in a manner that mimics
biological relevancy remains a challenge.
At a minimum, for the hit-to-lead process to be successful,
the assay systems employed to evaluate compounds should provide a clear
understanding of compound affinity for the intended target, as well as a clear
“profile” of compound specificity, which is typically thought of as a
measurement of the affinity of the compound for its intended target relative to
the affinity of its interactions with other, often related, targets. Both of
these properties can contribute to the ultimate success of a compound: Those
that bind tightly (have high affinity for their intended target) can in theory
be used at lower concentrations, and those that bind selectively should by
definition have fewer off-target effects, which can contribute to side effects
and toxicity.
While compound affinity for the target of interest can often
be assessed using the same assay system used to initially identify and/or
characterize the compound, specificity is often assessed by performing assays
against a larger number of targets. Because of the scale and complexity of this
profiling and a requirement for standardization, nuances may be lost during the
profiling process.
For example, in the case of kinase-directed compounds,
profiling is often performed against panels containing the active forms of
kinases. While this simplifies the assays being performed (it is easier to
measure the activity of an enzymatic reaction when the enzyme is active), it
removes the nuance of compounds that may bind preferentially to the
non-activated form of a kinase, and thereby stabilize the non-active state,
which may prevent further activation.
Additionally, although more than 500 protein kinases exist
in the human genome, methods to express and/or assay all of these kinases do
not exist, and even the broadest panels lack full kinase coverage. The lack of
full target family coverage and lack of easy control over kinase activation
state is compounded by the fact that for both technical and economic reasons,
full-length targets are often not used (catalytic activity may be assessed
using only the catalytic domain of the kinase), and the targets are often
expressed in non-mammalian systems or as domains expressed on the surface of phage
particles.
In addition to target affinity and specificity, a third (and
less commonly appreciated) property of a compound that can correlate with both in
vivo efficacy as well as safety is often
referred to as drug-target residence time. This property is related to the
average time that the compound remains associated with its intended target
before dissociation.
This leads to a second corollary that can be associated with
residence time, that being an increase in the “effective” selectivity of a
compound. For example, a drug may bind to multiple targets, but if the
“off-target” events have short residence times such that the drug may be
eliminated before these off-target events are detrimental, then side effects
and toxicity may be lessened.
The importance of drug-target residence time has been
recognized for many decades, and at least via retroactive analysis, there are
numerous examples where the in vivo
properties of a compound or set of compounds can be explained or rationalized
based upon residence times. Despite the recognized importance of drug-target
residence time, measurement of this property is not commonly performed when
prioritizing compounds early in the drug discovery process. While affinity can
often be measured using fairly standard methods, and first-pass specificity can
be determined by profiling compounds against an appropriate panel of related
targets, compound residence time is often measured using complex kinetic
experiments or in systems using immobilized targets.
For example, a traditional enzymatic method for measuring
the rate at which a compound dissociates from a target is to first incubate the
target and the drug at a concentration above the Kd value for that interaction,
to rapidly dilute the sample into assay buffer such that the total
concentration of drug is now below the Kd value, and then to measure the
initial rate of a catalytic reaction at various time points after the dilution
has been performed. As the compound dissociates from the target the enzymatic
activity is restored, and the rate at which this restoration is seen can then
be correlated with residence time.
Although conceptually simple, the dissociation process
cannot be monitored in real-time, and multiple catalytic reactions
(experiments) are required to determine residence time for a single compound.
Alternative non-activity based assays can be performed in a similar format
using radioactive probes that bind to the active site as compound dissociates,
and bound radioactivity can be measured after a separation step is performed
(to remove unbound radioactivity), but these methods come at the regulatory and
safety expense associated with the use of radiation.
An alternative approach to measuring compound dissociation
is by surface plasmon resonance (SPR) or similar optical methods in which the
target is immobilized (attached) to a surface, and compound binding (and
dissociation) can be measured in real-time as solutions containing compound are
passed over the immobilized target. Limitations on this approach can include an
appropriate level of sensitivity, as the signal is dependant in part of optical
changes induced by a small molecule (ca. 500 Daltons in molecular weight)
binding to a much larger protein (ca. 50-100 kilo Daltons), which can be
extremely small. However, due to the flexibility of the technique (which
includes no requirement for labeling either the target or the receptor with any
sort of “tag” or handle) and the rapid pace at which technological improvements
are being made, it is expected that these types of measurements will push into
routine use at earlier stages in the drug discovery process in the future.
Recently, several techniques were described at the 2010
Society for Biomolecular Sciences (SBS) conference in April in Phoenix, from
both my own group as well as scientists at GlaxoSmithKline, that combine
certain elements of existing approaches in an attempt to develop a homogenous,
real-time assay system for measuring drug-target residence time. As with the
traditional radiometric approach to measuring compound off-rates, labeled
compounds that can bind to a target active site as a drug dissociates are used,
but in these cases the labels used are fluorescent rather than radiometric.
Dr. Kurt Vogel is director of R&D within the discovery and ADMET
systems segment of Life Technologies in Madison, Wis. In this role, he has lead
teams focused on the development and commercialization of products and services
aimed at early-stage drug discovery, particularly those involving
fluorescence-based readouts and formatted for high-throughput screening
applications. Back  |
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