The Application of Label-free Technologies in Drug Discovery for Soluble Protein Targets

August 01, 2011

Abstract

A vast number of technologies are currently available for the development of assays for soluble protein targets that are high throughput screening compatible. These assays are usually developed in microtitre plate format so that large numbers of compounds can be evaluated in a timely manner. In addition, for many assays, various components need to be labelled so as to allow target activity to be monitored. Upon completion of a screening campaign, the activity of a selection of hits are confirmed in a variety of secondary assays, some of which may be label-free and thus potentially of more physiological relevance. In this context, the successful application of label-free assays for the kinase, protease and histone deacetylase class of soluble enzyme targets will be discussed. Notable applications of these label-free assays include HPLC, isothermal titration calorimetry, surface plasmon resonance and NMR. In addition, the application of pseudo-label-free assays, in which the key biological event occurs in a label-free environment but the detection of target activity requires a label will also be discussed.

Introduction

The discovery and development of small molecule therapeutics to treat diseases typically requires ten years of research and significant financial investment [1]. Despite the scientific expertise of the research teams involved in drug discovery programs, the attrition currently associated with the progression of compounds is significant and financially unsustainable [2]. A major issue to date is that an excessive number of compounds discovered for drug discovery purposes fail to demonstrate safety and efficacy in appropriately controlled clinical trials [3]. In light of this, significant efforts are underway within industry and academia to increase the likelihood of compounds progressing in the drug discovery value chain [4-8].

Small molecules currently form a large part of the therapeutic agents approved by the regulatory authorities to treat diseases and this trend is likely to continue in the future [9]. This view is further reinforced when considering that currently there are no shortage of targets available to explore for drug discovery purposes in the postgenome sequencing era, for example, there are more than 500 known kinases but only a small fraction of these are being explored for drug discovery purposes [10]. In many cases, clinical candidates that have been developed for the kinase target class have failed to demonstrate efficacy in disease patients despite their optimized potency and defined mechanism of action. A major reason for the lack of efficacy is that the targets against which the small molecules were developed lacked sufficient validation and the use of inappropriate animal models to benchmark small molecules prior to their evaluation in humans [11].

High Throughput Screening for Soluble Protein Targets

High throughput screening (HTS) has been a useful method for identifying compounds that can be starting points for drug discovery [12-13]. Although biochemical assays have historically been the most amenable to this method of drug discovery, cell-based assays are becoming the method of choice for screening activities [9]. These cell-based assays are often phenotypic in nature and it is advantageous to have in place a biochemical assay in which compounds can be evaluated so as to confirm that they directly modify the activity of the target or bind to it. In addition, it can be difficult to drive medicinal chemistry and structureactivity-relationship (SA R) in the absence of the knowledge of the target(s) upon which the compounds act. Therefore. the ability to evaluate compounds in target-based assays is important as it adds confidence that any measured activities are genuine and not assay format specific artefacts [14].

Defining a Small Molecule Screening Compatible Label-free Assay

The term “label-free” assay has a relatively broad meaning and it is important to be aware of their limits. It implies that this type of assay offers an improvement upon an assay that makes use of a “label” however, this may not be the case. The primary aim of small molecule screening in drug discovery is to discover chemical starting points for the development of a Lead, and subsequently, a Candidate molecule. The progression of the initial hits will require significant improvements with regards to their potency against the primary target, selectivity and liability profiles, ADMET and physicochemical properties [15]. Many label-free assays for soluble protein targets make use of substrates that are not physiological and therefore these are potentially as artificial as assays that require labels. It is therefore important to consider pseudo-label-free assays in which the key biological event e.g., substrate phosphorylation (by a kinase) or substrate degradation (by a protease) make use of the native target and substrate which are in essence label-free, but the detection of the extent of phosphorylation or cleaved protein are made using methods which make use of labels and these are described below with respect to the kinase, protease and histone deacetylase class of enzymes.

The Development of Label-free Assays for Protease Enzymes

The recognition characteristics of protease enzymes has been established using both chromogenic and fluorogenic substrates [16,17] as well as more sophisticated approaches and these include label-free approaches. Fluorescence resonance transfer (FRET) methodology has extensively been used [18] and make use of physiological substrates (i.e., relatively large proteins) that contain all of the potential molecular recognition sites often lacking in smaller peptide substrates. Cleavage of the protein substrate results in the generation of a product with an epitope that is recognized by a labelled antibody. Other approaches use mass spectrometric or HPLC analysis [19] or calorimetry when catalytic reactions are associated with relatively large heat changes [20].

The Importance of Label-free Assays Exemplified by Histone Deacetylase Enzymes

This is an important target class of enzyme for a variety of disease indications but most notably cancer [21], evidenced by the regulatory approval of two drugs, namely vorinostat which exhibits broad activity against histone deacetylase (HDAC) Class I and II enzymes and the natural product romidepsin which targets the Zn2+ dependent HDAC enzymes [22,23]. The initial approaches employed to monitor HDAC enzyme activity made use of radiolabelled histones as the substrate [24] and these assays are not the preferred option for high throughput screening. As in the case of protease enzymes, artificial substrates e.g., a peptide containing an ε-acetylated lysine that is C-terminally coupled to 4-methyl-coumarin-7-amide has been utilized as a substrate [25]. However, the performance of this assay for SIRT1 has been questioned by independent studies [26,27]. This included a comparison using a label-free HPLC assay that showed that deacetylase activity was only observed with the labelled substrate. Subsequent ELISA and Western blot studies using native full-length Ac-Lys383-P53 further confirmed the lack of activity for resveratrol in addition to biophysical studies using NMR, surface plasmon resonance and isothermal calorimetry confirming that the compound interacted directly with labelled-peptides in a dose dependent manner, however, the native peptides did not, thus illustrating the importance of label-free assays.

The Development of Pseudo-label-free Assays for Kinase Enzymes

These enzymes are implicated in a range of disease states and a number of small molecule inhibitors have been developed and approved for clinical use, particularly in cancer [28]. Although radioactive kinase assays have been extensively utilized in kinase drug discovery, these have generally been replaced by fluorescence based methods such as fluorescence polarization which rely upon binding of a fluorophore labelled-compound that binds the protein e.g., ATP [29], FRET which make use of a quenched peptide substrate that undergoes phosphorylation with the residual substrate undergoing proteasemediated cleavage and relaxation of the quenching [29], time-resolved fluorescence resonance energy transfer (TR-FRET) which make use of a labelledsubstrate which upon phosphorylation can bind a phospho-specific antibody [29]. Additional kinase assays making use of fluorophore labelled-peptides which can undergo phosphorylation such that they become more negatively charged relative to the unphosphorylated substrate can be exploited during electrophoresis to physically separate them [29]. Pseudolabel-free kinase assays can make use of catalytically active kinase protein and its full length substrate, both of which are label-free in the Amplified Luminescence Proximity Homogeneous assay which is an antibody-based proximity assay [30].

Conclusion

The primary aim of high throughput screening for drug discovery purposes is the identification of compounds that can be optimized to yield Lead and Candidate molecules for evaluation in clinical trials. Label-free assays have successfully been implemented to validate hits from high throughput screening campaigns as illustrated by the protease, kinase and histone deacetylase enzymes.

It is recommended that a variety of assays (in a range of formats) should be developed in parallel with each being compatible with high throughput screening. Subsequent to this, a subset of compounds from a small molecule library should be screened in all available assays and the hits correlated to identify overlapping populations as well as assay-specific false positives/negatives. After a thorough analysis of the output is performed, the assay that is likely to deliver compounds that will progress to the next stages of drug discovery should be utilized in the screening campaign itself.

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