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Case Study · cBTE Platform · Oncology

DEL Screening of a Difficult Protease Cancer Target: 11 Confirmed Inhibitors from a Single cBTE Screen

Using Cellular Binder Trap Enrichment (cBTE) against a 500-million-compound DNA-encoded library, Vipergen identified four chemical series and confirmed 11 binders with single digit micromolar KD-values against a protease widely regarded as one of oncology’s most challenging targets. Most of the binders were shown to be inhibitors.

500M

Compounds Screened

1

Round of selection

78

Primary hits

11

Confirmed binders

The Challenge

A high-value cancer protease resisting conventional inhibitor discovery

Proteases are implicated across a broad range of solid tumors, and the target in this study plays a role in multiple cancer types including lung, breast and colorectal cancer. Despite clear therapeutic rationale, it is well known as a very difficult target: only a handful of high-potency inhibitors have been reported to date.

The reasons are structural. The protease presents shallow interaction interfaces, ill-defined catalytic pockets, and intrinsically disordered structural elements that frustrate structure-guided design. Compounding this, the protein shows indications of instability and degradation, and screening in vitro on purified protein afforded 0 hits. 

Targets with these features — flat, flexible, and partially disordered — are exactly where traditional screening methods tend to underperform. What the program needed was a screen that interrogates the protein in a native-like and physiological relevant environment. Learn more about intrinsically disordered proteins as drug targets and enzymes as drug targets.

The Approach

One screen, inside living cells, against 500 million compounds

Vipergen screened the protease against Lib044, a YoctoReactor®-generated DNA-encoded library of approximately 500 million compounds (average MW ~610 Da).

Selection was performed using cellular Binder Trap Enrichment® (cBTE), Vipergen’s proprietary technology and the first and only method for screening DNA-encoded libraries inside a living cell. The DEL is microinjected into living Xenopus laevis oocytes in which the protease is expressed and specifically DNA-labelled, so that binding takes place in the crowded, physiologically relevant environment of the cytosol. Genuine binders are trapped together with the protease while non-binders remain unassociated and contribute only low-count background. For a target flagged for instability and degradation, this native-like format is a decisive advantage. 

Critically, cBTE requires no purified target protein. For a protease flagged for instability and degradation, this is a great advantage: The difficult purification step that would otherwise risk denaturation and artefacts is removed entirely, and hit recovery is driven purely by target affinity under native-like conditions. See how it compares in DELs vs. fragment-based screening and DELs vs. traditional HTS.

The workflow proceeded in four stages:

  • In-cell delivery. The 500-million-compound DEL was microinjected directly into living oocytes expressing the protease, hereby bypassing any membrane-permeability barrier.
  • Equilibrium binding & trapping. Compounds reached binding equilibrium with the target in the native intracellular environment, and genuine target–ligand binders were trapped together with their target.
  • Sequencing & decoding. Recovered library DNA was PCR-amplified, sequenced, decoded and counted to reconstruct how many times each compound was observed.
  • Thresholding. A mathematical threshold of 6+ observations was applied to the resulting signal plot, cleanly separating hits from random background and yielding 78 primary hits.

Figure 1.

cBTE signal plot for the protease screen. The number of compounds is plotted against the number of times each was observed by sequencing. High-count binders (green) separate cleanly from random background (grey). Applying a 6+ observation threshold yields 78 primary hits.

Let’s discuss your next discovery program.

From difficult enzymes to protein–protein interactions, transcription factors and intrinsically disordered targets, Vipergen’s cell-based DEL platform is built to deliver genuine, functional hits — fast. Get in touch to scope a screen against your target.
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The Result

Four chemical series and 11 confirmed inhibitors — from a single selection

The 78 primary hits were analyzed as a 78×78 similarity heatmap based on Tanimoto similarity, then organized using hierarchical clustering to reveal structural relationships between compounds. This clustering resolved four distinct chemical series (highlighted as blocks 1–4 in Figure 2), giving the program several independent starting points rather than a single, fragile chemotype.

Compounds were resynthesized and characterized, confirming 11 positives concentrated in the major chemical series 4, with binding affinities (KD) in the single digit micromolar range. Most of the confirmed positives were further shown to be inhibitors against the target. Hereby the developed compounds could both be amenable for inhibitor development or be used for targeted protein degradation by developing them into PROTACs. 

Figure 2.

Tanimoto-similarity heatmap of the 78 hits (78×78 matrix), sorted by best-neighbour method. Darker red indicates higher pairwise similarity (Tc); four chemical series emerge as distinct blocks (1–4). The right-hand KD track (green scale) maps measured affinities onto the clusters.

What this demonstrates

Functional hits identified in a single round of screening

This case illustrates what a native-like, in-cell DEL selection can deliver on a target defined by shallow interfaces, poorly formed pockets, disorder and instability, which typically challenges other hit identification methods. Working from binding affinity alone, cBTE surfaced multiple independent chemical series and functional inhibitors in a single round, with no prior structural bias required.

Key takeaways for your program

  • Difficult targets are tractable. Disordered, unstable or poorly pocketed proteins that resist HTS and immobilised DEL formats can still yield confirmed inhibitors under cBTE.
  • Series, not singletons. Four independent chemical series give medicinal chemistry robust, parallel starting points and reduce single-chemotype risk.
  • Single-round efficiency. A 500-million-compound library was interrogated in one selection, with a clean 6+ threshold separating genuine hits from background.
  • Function, not just affinity. Most confirmed positives are inhibitors, giving a direct path toward functional lead series.

Explore Vipergen’s cell-based DEL discovery services, our broader services overview, and cancer drug discovery at Vipergen — or read more on understanding DEL screening from target binding to hit validation.

Do you have a difficult target?

Contact Vipergen to discuss screening your protease, enzyme or intrinsically disordered target with cBTE. Get in touch.

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