The Deconvolution Revolution: Strategies for Target Identification

In the drug discovery and development process, researchers aim to identify a compound that is active against a specific molecular target. This compound is then iterated upon to optimize its pharmacokinetics, deliverability, and other key parameters, ultimately generating a drug that can be evaluated in pre-clinical models and clinical trials. In one approach – known as target-based drug discovery – researchers start with a known molecular target, then seek to identify chemical compounds that interact with this target of interest. Downstream assays further elucidate the mechanism of interaction and validate the functional effects of target binding. A second drug discovery framework – phenotypic drug discovery – takes a different approach. In phenotypic drug discovery, researchers assess chemical content for its ability to evoke a desired phenotype (such as cellular differentiation, induced expression of a reporter gene, or cell death). Once a promising molecule has been identified, further research is done to determine its mechanism of action, including the specific cellular target(s) through which it functions. While both strategies are in common use, some experts suggest that compounds discovered through phenotype-based techniques may be more efficiently translated into clinical innovations, as the screening methodology more accurately reflects the complex biological context in which these drugs must act.

What is target deconvolution?

Target deconvolution refers to the process of identifying the molecular target or targets of a particular chemical compound in a biological context. As such, it is a key component of phenotypic drug discovery workflows, where it is used to clarify the mechanistic underpinnings of hits that emerge from initial screens. Following the identification of a promising compound by phenotype-based screening, researchers can leverage target deconvolution strategies to clarify the molecular targets of the compound. This endeavor can encompass the elucidation of both on- and off-target interactions, and associated efforts may also seek to identify affected signaling pathways or other cellular disruptionsdownstream of primary target binding. Target deconvolution provides a critical link between phenotype-based screening assays and the subsequent stages of compound optimization, mechanistic interrogation, and preclinical characterization.

How does target deconvolution work?

A wide range of techniques can be used for target deconvolution, each best-suited for a particular set of applications or targets. By utilizing an appropriate assay or combination of assays for target deconvolution, researchers can most effectively determine the mechanism of action underlying hit activity. Broadly, target deconvolution experiments fall within the category of chemoproteomics, meaning that they interrogate interactions between proteins and bioactive small molecules. Canonical target deconvolution strategies involve the development of a chemical probe that links the small molecule of interest to some type of handle, thereby enabling bound targets to be captured and identified. However, label-free target deconvolution approaches have also been developed, providing significant utility in cases where compound labeling is disruptive, technically challenging, or otherwise infeasible.

What experimental approaches can be used for target deconvolution?

One major class of target deconvolution strategies leverages affinity-based chemoproteomics to isolate and identify target proteins. In this approach, a compound of interest (e.g., a chemical hit from a phenotypic screen) is first modified so that it can be immobilized on a solid support, then exposed to cell lysate. Proteins that bind to the immobilized ‘bait’ can then be isolated through affinity enrichment and characterized by mass spectrometry. This assay not only reveals the cellular targets of a compound under native conditions but can also provide dose-response profiles and IC50information, guiding downstream drug development efforts. This technique works well for a wide range of target classes and is often considered a ‘workhorse’ technology. However, its use for target deconvolution does require a high-affinity chemical probe that can be successfully immobilized. Researchers seeking to harness this approach can do so through the commercially available TargetScout service, which offers flexible options for robust and scalable affinity pull-down and profiling.

A second class of target deconvolution strategies relies on (re-)activity-based labeling to identify cellular targets. Based on the principals of activity-based protein profiling (ABPP), these strategies employ bifunctional probes containing both a reactive group and a reporter tag. Probes are provided to cells or lysates and covalently bind to molecular targets, labeling target sites such that they can later be enriched and identified via mass spectrometry. In one iteration of this technique, an electrophilic compound of interest is functionalized and its binders identified directly. In a second variation, samples are treated with a promiscuous electrophilic probe with and without the compound of interest; targets are identified as sites whose probe occupancy is reduced in the presence of the competing compound. This approach is powerful but requires the presence of reactive residues in accessible regions of the target protein(s). Researchers interested in using this technology for target deconvolution can employ CysScout, which enables proteome-wide profiling of reactive cysteine residues, or customized assays based on non-cysteine probes.

Target deconvolution can also be accomplished through photoaffinity labeling (PAL). In this approach, a trifunctional probe is comprised of a small molecule compound of interest, a photoreactive moiety and an enrichment handle. Following binding of the small molecule to target proteins in living cells or cell lysates, light exposure induces the formation of a covalent bond between the photogroup and target. The handle is used for the enrichment of interacting proteins, which can then be identified via mass spectrometry. PAL probes can be designed with different photoreactive groups and with diverse group positions, enabling their physiochemical properties to be optimized for desired analyses. While it may not be suitable for targets with shallow surface binding sites, this approach is particularly useful for the study of integral membrane proteins, as well as to identify compound-protein interactions that may be too transient to detect by other methods. Photoaffinity labeling services for target deconvolution include PhotoTargetScout, which encompasses both assay optimization and target identification modules.

While the preceding target deconvolution approaches require incorporating a label or handle component into the compound of interest, other techniques have been developed to enable label-free target identification. Label-free strategies can be particularly valuable because they enable compound-protein interactions to be evaluated under native conditions, without the need for chemical modifications that may disrupt the compound’s conformation or function. One approach for label-free target deconvolution– solvent-induced denaturation shift assays – leverages the changes in protein stability that often occur with ligand binding. By comparing the kinetics of physical or chemical denaturation before and after compound treatment, researchers can identify compound targets on a proteome-wide scale. This technique can be challenging for very lowly abundant proteins, very large proteins, and membrane proteins. For feasible targets, however, this technology can provide invaluable insights into chemical interactions in a physiologically relevant context, advancing target deconvolution and off-target profiling. Researchers interested in applying this strategy to their own compounds of interest can do so via SPICE, a commercially available in-cell proteome-wide protein stability assay.

How can target deconvolution advance drug discovery and development?

Target deconvolution strategies play a critical role in the drug discovery and development pipeline, bridging the gap between initial discovery screens and downstream research efforts. By identifying the on- and off-targets of a chemical compound, researchers can evaluate its feasibility as a drug candidate and elucidate its mechanism of action. This is especially critical in phenotype-based screening frameworks, which identify hits based on their ability to induce a desired cellular phenotype rather than by their ability to bind a designated target protein. Once target deconvolution has been performed, researchers are empowered to optimize a drug candidate to enhance on-target activity, reduce off-target activity, improve deliverability, and more. By partnering with an experienced target deconvolution service provider, researchers can ensure that they select the most appropriate experimental approach for their application and that their target deconvolution assays are performed accurately, efficiently, and reliably.

To learn more about target deconvolution and explore how these technologies can accelerate your research program, get in touch with our scientific team.

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