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Harnessing the Versatility of Optical Biosensors for Target-based Small-molecule Drug Discovery Tim Patrick Kaminski, Anders Gunnarsson, and Stefan Geschwindner ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00735 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 18, 2016
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Harnessing the Versatility of Optical Biosensors for Target-based Small-molecule Drug Discovery Tim Kaminski1, Anders Gunnarsson1 and Stefan Geschwindner1* 1
IMED Biotech Unit, Discovery Sciences, AstraZeneca Gothenburg, Pepparedsleden 1, 43183 Mölndal, Sweden.
KEYWORDS: drug discovery, small molecule, kinetics, single molecule, surface-plasmon resonance, waveguidegrating, bio-layer interferometry ABSTRACT: Optical biosensors entered target-based small-molecule drug discovery more than two decades ago and have since transformed into a value-adding component in the decision-making process. Here, we briefly highlight the major application areas of optical biosensors and focus on desirable profiles of such platforms in order to ensure their effective use in small molecule drug discovery. Furthermore, we will emphasize current technology-based constraints and discuss experimental strategies to address these limitations as well as provide a view of necessary technology improvements for next generation platforms.
Current Use and Impact of Optical Biosensors in Target-based Small-molecule Drug Discovery Optical Biosensors have seen widespread application in target-based drug discovery following the appearance of the first commercial platforms in the early 90´s.1-2 Instruments based on surface plasmon resonance (SPR),3 optical waveguide grating (OWG)4 or biolayer interferometry (BLI)5 are predominantly applied in early drug discovery focusing on small-molecule screening and profiling, antibody characterization and selection in addition to biomarker detection, of which the latter two will not be discussed here. The major application areas in the small molecule space aim to identify and characterize the interaction of compounds with their target protein, which encompass (i) primary screening, (ii) validation of target engagement, (iii) identification of the molecular mode of action (MoA) along with (iv) detailed characterization of compounds binding such as binding kinetics and thermodynamics. A summary of established and currently emerging biosensor platforms that are used in the context of small molecule drug discovery is provided in Table 1, including their primary application areas as well as the associated information content. Primary Screening Primary screening using optical biosensors can utilize plate-based OWG systems to enable screening of
relatively large compound libraries (>10 000). The experimental flexibility allows equally for a biochemical4 or a cell-based readout6, with the notion that the later rather reflects the consequences of compound binding through changes in cell morphology. In contrast, Fragment-based drug discovery (FBDD) requires screening of much smaller compound libraries (1000 data points per day.
Figure 1. The 3 R´s of biosensing representing key factors for successful generation of biosensor-derived data to support target-based small-molecule drug discovery: Right throughput, right sensitivity and right reagent.
Once moving forward in this process, an ideal scenario is the use of the same or a similar biosensor platform and/or assay configuration to continuously support the maturation of weak binding fragments into more potent
chemical leads. Simultaneously, one often needs to enable hit validation originating from alternative hit finding approaches such as HTS. Consequently, such platforms need to be able to cover a broad affinity range from mMdown to pM-binding affinities. This will ensure such platforms are applicable throughout the early drug discovery process from hit finding and validation via lead identification into lead optimization and beyond. A key but often neglected aspect is the stringent requirements on biosensor-compatible protein reagents since they make intimate use of the transducer surface. Hence, one of the most critical aspects of data quality is the ability to tether a functional target with high stability to the surface. In addition, the density of tethered functional target must be relatively high, since the signal amplitude scales with the surface density of the tethered ligand. This is particular critical in the case of small molecule interactions due to the relatively low signal generated upon binding. In summary, optimization of protein reagent is key for successful deployment of any biosensor platform in early drug discovery. There are a number of intrinsic and extrinsic factors that will influence this optimization workflow. Target stability can be affected by the buffer and its supplements, making screening for the right assay conditions often a prerequisite. Frequently, the manipulation of the target protein itself is critical for success. This may include addition of short peptide tags (e.g. Avi- or hexa-His) to allow for directed and thus more controlled target tethering in contrast to non-directional covalent- (e.g. amine or maleimide chemistry) or non-covalent (charge, hydrophobic) tethering approaches. For more challenging targets such as membrane-bound receptors, iterative rounds of point mutations have sometimes been successful.19 Whilst many native receptors are unstable and rapidly denature at the sensor surface, thermostabilized mutants of the same receptors can retain their functionality and stability in a surface-tethered configuration for a number of days. Another commonly employed strategy is the use of truncated proteins containing only the domain of interest that is targeted with small molecules. In such cases, it is very important to closely monitor that those variations and alterations do show similar ligand binding patterns as to the native protein. Observed discrepancies could indicate a change in pharmacology when going into an in vivo model to test the target hypothesis and thus need to be addressed. Nevertheless, such modifications are typically tedious and tend to be very resource-intensive without guarantee for success. In summary, the throughput and the sensitivity of any biosensor platform typically provides a framework for defining optimal protein reagents or tools in order to meet the individual project requirements. These can vary quite substantially and thereby expose both strengths as well as weaknesses of the individual biosensor platforms used in drug discovery.
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Strengths and Weaknesses of Main Biosensor Platforms in Drug Discovery The choice of optical biosensor platform is heavily dependent on application and assay requirements. As plate-based biosensor platforms like OWG offer a similar throughput as biochemical assays used in HTS, they provide an attractive alternative for biophysical screening. Nevertheless, those platforms typically experience relatively low sensitivity when monitoring direct and specific small molecule binding, which is in part a consequence of the limited tethering capacity of the functionalized biosensor surface. Thus, such platforms are more useful when trying to identify so called “promiscuous” or aggregation-based inhibitors, as those signals tend to be much larger and are easily detectable.20 Changing the assay configuration can in some cases bypass the sensitivity limitations of OWG platforms and enables the screening of weakly binding fragments to support FBDD. This is achieved through directed tethering of a functionalized tool compound, which still retains good binding competence to the respective target protein. Challenging the modified biosensor with the target protein typically leads to a detectable signal, as the amplitude scales with the change in molecular mass on the biosensor. This signal can be subsequently modulated by compounds that compete for the same binding site in solution, thus this assay configuration is also referred to as inhibition in solution assay (ISA).21 A different picture emerges for SPR-based instruments. Whilst OWG platforms provide much higher throughput than sequentially operating systems (interrogating one compound or concentration at a time), SPR systems typically offer much higher sensitivity at the cost of lower throughput. This generally makes it the platform of choice for driving FBDD campaigns in a direct binding assay (DBA) configuration, as this allows reliable detection of weak interactions with small molecules to surface-tethered proteins.7 As a consequence of the fluidics-based nature of these platforms, there is the additional possibility to record binding kinetics data. Whilst plate-based systems typically operate at equilibrium, which only allows for affinity (Kd) determination, fluidics-based systems such as SPR also provide the opportunity for kinetic readout (kon, koff) and thus offers higher information content. However, even for platforms with the same optical transducer, e.g. SPR, the balance between sensitivity and throughput is crucial. Although “regular” SPR is unrivaled in terms of sensitivity, imaging SPR22 offers accelerated throughput but at the expense of sensitivity, thus making the latter technology less attractive to small molecule research. In order to address the sensitivity issue, some SPR imaging platforms are making use of chemical microarrays, which have shown to be particular useful for fragment screening.23 Whilst following similar principles for sensitivity enhancement as the ISA, kinetic information is more challenging to assess in this assay configuration. Whereas throughput and sensitivity in addition to robustness has continuously improved over time, all biosensor platforms suffer from different limitations that
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are intrinsic to their build up. Besides the sensitivity limit that is dictated by the molecular mass of the interactants, also throughput can be surprisingly affected by systemrelated factors like binding kinetics. As optimized compounds gain dramatically in potency by several orders of magnitude once progressed from an initial hit towards a candidate drug, they frequently display residence times of hours or even days. For a reliable determination of binding kinetics, the measurement time thus needs to be extended24 and a complete dissociation is required to present a compound-free biosensor for subsequent experiments. Even with throughput-enhancing strategies like single-cycle kinetics25 it can easily take 12-24h to obtain affinity and binding kinetics data for a single compound. BLI can address some of those limitations, as it elegantly combines some advantageous features of SPR and OWG by offering a plate-based readout with the option for kinetic measurements.26 A range of biosensor-tips (8-16) are dipping into an analyte solution that is presented in a plate that can be agitated to mimic a fluidics-based scenario for kinetic measurements. Those sensors can be discarded afterwards to enable a new round of measurements, thereby eliminating the need to wait for complete compound dissociation. This strive for parallelization has also seen some recent advances in the development of throughput-enhanced SPR platforms, enabling 8 analyte evaluations in parallel. Despite extensive optimization of protein reagents and assay conditions, it can sometimes be challenging to make a clear-cut assessment of the binding specificity. Whilst it is straightforward to identify compounds that deviate from the expected binding behavior in terms of saturable, reversible binding following a 1:1 binding model27, this is often much more difficult to achieve for compounds that specifically engage with the target. In this case, one is forced to conduct additional experiments to look for competition with known, well-characterized tool compounds or to perform a subsequent ISA experiment as previously described. This obviously requires readily access to such tool compounds, which preferably display high potency and/or can be modified to accommodate a suitable linker for directed tethering. Both approaches come at the cost of additional reagent consumption and extended timelines for decisionmaking.
Next-generation Biosensor Platforms addressing Drug Discovery Needs One common denominator in the experimental workflow of the discussed biosensor platforms is the need for two separate experiments. First, the sensor surface is brought into contact with the analyte. Thereby, the convoluted association and dissociation reaction is measured. Typically, this reaction is observed until equilibrium coverage of the sensor surface is reached. Subsequently, the sensor surface is exposed to an analyte-free solution to monitor the dissociation reaction alone. The possibility of simultaneous quantification of association- and dissociation-rates would obviously represent a great ACS Paragon Plus Environment
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technology enhancement, which can be realized with single molecule biosensor platforms. Such novel biosensor platforms and their potential value for drug discovery have recently been described by making use of the ISA configuration.28 Whilst the tool compound is tethered to the biosensor surface, the target protein is attached to or incorporated into the lipid layer of fluorescently labeled liposomes. Using a standard TIRF microscope, the binding of the target-liposome complex to the surface can be monitored with single molecule resolution.With each liposome containing at maximum a single copy of the target protein, each observed binding event reflects the interaction of a single target protein with a single tool compound at the surface. The ability to monitor each binding and release event at the surface under equilibrium conditions allows for an estimation of association and dissociation rates from a single experiment without micro-fluidics or alternative liquid handling.29 This has many practical implications such as unrivaled sensitivity and thus provides a good rationale to eventually succeed existing biosensor platforms. The requirements on the tethered tool compounds are less stringent, as low affinity is not only tolerated but often even desirable, as it increases the number of observable binding events in a given timeframe due to faster dissociation. Additionally, the use of tool compounds in conjunction with single molecule sensitivity furnishes the opportunity to distinguish specific from non-specific binding events, as they will show different kinetic profiles that can be deconvoluted. This is essentially impossible with technologies that are based on ensemble measurements like SPR, OWG or BLI. Furthermore, the use of liposomes offers the possibility to study ligand binding even to labile membrane proteins in an almost native-like environment, without the need of extensive protein purification or stability-enhancing mutations.29 This strategy does not exclude soluble proteins, as they can be tethered to liposomes following similar strategies as for traditional biosensor experiments. Finally and most importantly, performing experiments at the single molecule level has surprising consequences for the limit of detection and thus the sensitivity. By increasing the observation time one is able to detect more binding- and release-events to the biosensor. Therefore, the lower limit of detection is constantly decreasing with increasing measurement time and is not a fixed and platform-dependent parameter anymore.
Conclusion and Outlook Over recent years, optical biosensors have been positioned as a vital part in small molecule drug discovery. The continuous improvements of existing technologies have expanded their application areas significantly. For example, state-of-the-art SPR systems have now reached a level of sensitivity that readily allows direct monitoring of small molecule interactions even at sub-optimal assay conditions (e.g. only a fraction of the tethered target is functional). However, new platforms are also starting to emerge in the drug discovery arena. One
example is single molecule microscopy, where evanescent-field illumination combined with surface functionalization provides a readout at the level of individual molecules. This provides unique opportunities such as the possibility to monitor binding kinetics at equilibrium in a plate-based format, thereby combining the benefits from plate-based (throughput) and flowbased systems (kinetics) in one platform. Another advantage lies in the extremely high sensitivity, which reduces protein consumption and allows measurements across a wide affinity range. This, together with the significantly reduced reagent consumption furnishes the opportunity to generate unique value for small molecule drug discovery.
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected] Author Contributions The manuscript is written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT We would like to acknowledge all colleagues within the global AstraZeneca Biophysics community for inspiration and their continuous strive to unleash the full potential and maximise the value of optical biosensors in drug discovery. Furthermore, we would like to thank the internal AstraZeneca post-doc program for the great support it has given to Tim Kaminski and Anders Gunnarsson. In addition, we want to thank Helen Boyd for input on the manuscript.
ABBREVIATIONS BLI, biolayer inteferometry; DBA, direct binding assay; FBDD, fragment-based drug discovery; HTS, highthroughput screening; ISA, inhibition in solution assay; OWG, optical waveguide grating; SPR, surface plasmon resonance; TIRF, total internal reflection fluorescence
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