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Guest Commentary: Prioritizing hits based on drug-target residence time
September 2010
by Dr. Kurt Vogel  |  Email the author

Amid the complexity and expense of the small-molecule drug discovery process, from identification and validation of a "drugable" target to the development of an understanding of the impact of pharmacogenomic differences in patient populations on drug action, lies the "hit-to-lead" process in which compounds that show activity in an assay system are iteratively improved upon through medicinal chemistry that is guided by more detailed assays and filtering criteria.  
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.
Given the fact that the majority of small-molecule drugs directed toward kinases presently target the ATP binding site, and that a plethora of proteins use ATP as a substrate (and therefore contain ATP binding sites), the ability of any profiling process that is limited to determining specificity against only kinase targets is clearly incomplete. Although elegant methods have been described that can identify the target of a small-molecule drug from within a cellular lysate (thereby exposing a compound to all possible binding partners present in a particular cell type), such methods still contain shortcomings and are difficult to implement in a cost-effective manner. 
Despite the recognized challenges of developing appropriate assays for moving compounds forward during the hit-to-lead process, and the shortcomings inherent in any of the available methods, this process remains crucial to the development of selective and efficacious drugs, with the important caveat being that the information that can be gleaned by any one method used in the process should be evaluated with a clear understanding of the limitations associated with that method.  
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. There are several reasons why this property can correlate with compound success. The first is intuitive: For a drug to be active against a target, it needs to be physically associated with that target. While association of a compound with a target is dependant on the concentration of the compound, the rate of dissociation is independent of drug concentration and is a property of the drug-target complex. Since any drug that is not associated with a target is available for metabolism, degradation or excretion, if a compound has a slower off-rate than these competing processes, it can remain efficacious for a longer period of time than if it had a shorter residence time.   
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. In either case, binding of the fluorescent probe to the target active site can be monitored in real time as the drug dissociates from the target by a change in fluorescence signal. Although in each approach there is a requirement to generate a fluorescent probe that will bind to the target of interest, for many target classes (such as kinases), there are well-described tool compounds that bind broadly across a target class, and can therefore be used to develop a small set of probes that could be used to determine residency time across a large number of related targets. Time will tell if these methods can be developed and validated to the extent that they may become a routine part of the lead optimization process to help develop better drugs faster.
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.



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