High-Throughput Optical Label-Free in Early Drug Discovery: From Biomolecular Interactions to Cellular Phenotypes
Julio Martin, Ph.D.
Molecular Discovery Research
GlaxoSmithKline
Abbreviations
GPCRs: G protein-coupled Receptors
HEK MSR-II: Human embryonic kidney cells stably expressing the macrophage scavenger receptor class II
HTS: High-throughput Screening
SPR: Surface Plasmon Resonance
RTKs: Receptor Tyrosine Kinases
The explosion of genomics in the 90's was expected to generate an abundant supply of new targets amenable to highthroughput screening assays. Some experts estimate that out of the genome sequencing, 40% of human genes have no known molecular function [1]. The conventional approach to drug discovery relies on the precise definition and formal validation of a few selected targets. By contrast, the chemical-genomics approach is based on genome-wide definition of many targets followed by drug screenings for most of them and complete biological validation of a few targets using tool compounds identified. The difficulty resides on the rapid definition of automated drug screening assays for such large numbers of proteins.
"Corpora non agunt nisi fixate" ("A drug will not work unless it is bound"), Paul Ehrlich
Most of the primary screens pursue compounds that modulate the function of a target protein. Assays and reagents are designed and produced in an ad hoc basis, which demands significant allocation of resources beforehand at risk of not knowing the actual tractability of the target. The universal principle of the action of drugs is the binding to their target. Screening methodologies looking at the binding of a compound to a given protein constitute a possible workaround.
"The prismatic qualities of the assay distort our view in obscure ways and degrees", James W. Black
No matter how complex, an assay will always imply reductionism. Relevance is linked to predictability and not to complexity of performance. Extrinsic labels and artificial biology (e.g., over-expression of proteins, chimeras, artificial coupling to signaling pathways and reporters) are usually engineered in order to probe the activity of the target. As a result, assay development is hampered and the biological texture of the target is altered for no longer resembling all features of a native environment. Thus, some assays may mislead the biological relevance of a particular compound. Compound testing in native cells that express endogenous receptors coupled to natural signaling pathways a priori will be a more relevant surrogate. Target tractability lies on precedent success. Novel experimental approaches can also help to increase the tractability of some target classes.
All assays are prone to interferences that can mislead the triage of compounds. The early identification of nuisances and promiscuous hits alleviates attrition rate, waste of resources and false expectations. Label-free assays can be exploited as orthogonal tools to confirm genuine responses of compounds.
Plate-based label-free biosensors coupled to optical detection are compatible with current screening technologies and infrastructure. They constitute a versatile platform that can accommodate a broad range of applications and contribute to fill some of the gaps aforementioned. This article will revise the basic principles of this technology, the state-ofthe-
art applications in early drug discovery, as well as some of the current challenges to face.
Advantages of Label-Free Methodologies
Since the first introduction of surface plasmon resonance (SPR) sensors in the early 1990´s, a variety of different label-free methodologies have been developed and adapted for use in basic research [2]. Expectations from label-free have risen exponentially within the scientific community over the last five years [3]. Amongst the main advantages of label-free detection, it
is worth remarking:
- Closer resemblance to native systems. Label-free removes the experimental uncertainty stemming from the effect of exogenous labels on the specimen of study (e.g., engineered cells, conformational restrictions and steric hindrances).
- Systematic and generic approach to assay development and screening. Prospect to faster turnarounds, less resources, lower cost and accessibility to reluctant targets.
- Non-destructive and real-time direct monitoring. On- and offkinetics and receptor de-sensitation can be measured. Also, the reuse of cells and proteins in sequential experiments is feasible.
The throughput of a biosensor system will determine its usefulness. Unlike biosensors embedded in a flow chamber, systems based on simple and disposable assay vessels (e.g., microwell plates) can provide an unsurpassed parallel processing capacity. Nowadays, there are at least two distinct technology platforms compatible with assays running in microplates and high-throughput mode. They differ in the biophysical principle of detection, namely electrical impedance or index of refraction. The fundamental advantage of the latter is that a direct electrical connection between the excitation source, the detection transducer and the sensor is not required. Consequently, design and fabrication become advantageously simplified and versatile. They offer high versatility of biological applications: from direct binding of small compounds to phenotypic cellular assays.
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