DNA-encoded libraries – DELs, for short – have attracted the attention of the pharmaceutical industry in recent years, promising the ability to readily screen millions or billions of potential ligands in parallel by linking small-molecule test compounds to unique DNA tags. The sheer magnitude of these screens provides obvious appeal. But with increased adoption has come increased recognition of the shortcomings of DEL technology. Before designing a DEL screen, it is important to consider some of the potential pitfalls associated with this approach, including target immobilization, library tagging, lack of quantification, resynthesis timelines, and library relevance. Below, we explore some of the critical drawbacks of DEL-based methods, highlighting the comparative advantages of Affinity Selection Mass Spectrometry (ASMS) as a high-throughput screening approach.

 

#1: Targets must be immobilized or tagged.

In a standard DEL screen, the target must be immobilized on a solid support, requiring the introduction of a tag or other modification. However, many clinically pertinent targets – such as membrane proteins – are prone to misfolding under these conditions. Even a ‘well-behaved’ protein can be structurally disrupted by tag addition, requiring substantial experimentation to confirm a suitable tag type and location. Protein-protein and protein-oligonucleotide complexes are especially difficult to immobilize successfully. Only one component protein can be directly tethered to the surface, and proper complex formation and stability must be painstakingly confirmed; these challenges are compounded for each additional protein in the complex.

Even once a protein (or complex) has been successfully immobilized, its ability to bind ligands is substantially impaired by steric hindrance. Portions of the protein that face the substrate are unlikely to be accessible, especially to large, tagged compounds (see #2). As a result, screens performed on immobilized targets are unable to capture the full range of ligand-target interactions. This framework is also prone to false positives arising from spurious interactions with artificial tags. In contrast, Affinity Selection Mass Spectrometry is label-free and can be performed in solution (as with the Affinity Ligand Identification System, or ALIS). This approach avoids the need for time-consuming protein engineering, requiring only minimal buffer optimization before the screening process can begin, and can be performed under physiologically relevant conditions.

 

#2: Test compounds must be tagged.

DEL technologies rely on the coupling of each test compound to a unique, PCR-amplifiable tag during the library synthesis process, allowing the identity of potential hits to be ‘decoded’ via next-generation sequencing (NGS). A typical tag can easily run from 50 to 70 bps in length, including a series of short variable identifiers as well as elements required for processing and sequencing, with an estimated molecular weight of ~40 kDa. Relative to an untagged small-molecule ligand, generally 0.1 to 1 kDa, a DEL tag is overwhelmingly massive – its addition can increase a ligand’s size by more than a hundredfold! Given the tight, cooperative nature of many target-ligand interactions (considered as “lock and key” mechanisms), the addition of such a bulky adduct is almost certain to restrict ligand orientation, occlude potential binding sites, and otherwise disrupt target-ligand interactions. Affinity Selection Mass Spectrometry, which screens untagged ligand libraries, avoids these obstacles altogether.  For target proteins that promiscuously bind DNA or interact with specific DNA sequences, DELs present a serious risk of false positives. This effect is particularly deleterious for RNA (and oligo-containing) targets, which are prone to complementary tag binding or other confounding oligo-oligo interactions. Affinity Selection Mass Spectrometry is not subject to these artifacts, making it well-suited to the screening of RNA targets and complexes with DNA or RNA components.

To learn more about using ASMS to identify RNA-targeting small-molecules, check out our blog post: Screening Strategies for RNA-Targeted Small-Molecule Drugs

 

#3: PCR prevents accurate quantification.

DEL approaches rely on polymerase chain reaction (PCR) to identify specific ligands from massively multiplexed pools. However, PCR does not amplify all products equally, limiting the quantitative capacity of DEL screens. As a result, any meaningful correlation between tag abundance and binding affinity is obscured, necessitating additional quantitative assays to effectively prioritize hits identified by DEL. PCR is also susceptible to stochastic dropout of low-abundance species, particularly in early rounds of amplification. In DEL screens, it is therefore impossible to distinguish a non-binding ligand from a ligand that binds but is not faithfully amplified. In contrast, Affinity Selection Mass Spectrometry identifies hit compounds based on molecular formula, rather than by a synthetic tag. As a result, comparative and quantitative binding information can be readily obtained and used to inform drug development decisions.

#4: Promising leads must be resynthesized and retested.

Once a DEL hit has been identified, the compound is resynthesized without the DNA tag, and its binding is retested using an orthogonal approach. In other words, hits from a DEL screen cannot be confirmed without invoking an entirely different assay for validation. This requirement reduces efficiency and delays experimental progress. In addition, DEL hits are commonly driven by spurious, tag-based interactions, or by intermediates and byproducts generated during library synthesis; significant effort can be required to clarify these false positives. In Affinity Selection Mass Spectrometry, targets are screened against an established library of commercially available and/or independently synthesized compounds which are immediately available to be validated.  Because compounds are identified by molecular formula, ASMS screening is not susceptible to false positives arising from impurities or breakdown products. The same platform can be used for initial screening, confirmatory screening, and further target interrogation, such as selectivity screening and competitive binding assays, minimizing lag time and accelerating drug discovery.

#5: Massive library sizes are misleading.

While DEL platforms enable billions of compounds to be screened per experiment, this increase in magnitude does not necessarily correspond to a more productive screen. A typical DEL is synthesized from a pool of chemical ‘building blocks’ using a combinatorial strategy, often based in click chemistry. This approach tends to generate planar molecules without significant 3D structure. What’s more, products are frequently inconsistent with Lipinski’s Rule of Five, making them poor candidates for drug development. DEL diversity is further affected by the use of DNA tags, which restrict all synthesis reactions to a narrow range of DNA-compatible conditions. These constraints significantly limit the structural diversity of screened ligands and reduce the functional power of DELs in identifying novel clinical leads. Affinity Selection Mass Spectrometry, in contrast, is performed using curated libraries of drug-like or lead-like compounds that are generally Rule-of-Five-compliant, increasing the chance of identifying developable, physiologically relevant hits.

 

Before embarking on a large-scale drug discovery experiment, it is important to carefully consider the advantages and disadvantages of different screening modalities. While DEL-based approaches exploit clever chemical syntheses to enable large numbers of potential ligands to be tested in parallel, they also present several major drawbacks. In many cases, the projected advantages of increasing screen size via DELs are counterbalanced by associated complications, such as the requirement to resynthesize and retest tag-free ligands, artificial screening conditions, and restrictions on functional library diversity. In contrast, Affinity Selection Mass Spectrometry can be performed under physiological conditions, screens curated libraries of pharmacologically relevant compounds, and does not require time-consuming resynthesis or orthogonal validation. When it comes to small molecule screening, more efficient hit-to-lead conversion can ultimately be achieved through the strategic application of Affinity Selection Mass Spectrometry to drug discovery efforts.

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