Petersen LK et al. (2021), Screening of DNA-Encoded Small Molecule Librari...

XB-ART-57808

J Am Chem Soc 2021 Feb 24;1437:2751-2756. doi: 10.1021/jacs.0c09213.

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Screening of DNA-Encoded Small Molecule Libraries inside a Living Cell.

Petersen LK , Christensen AB , Andersen J , Folkesson CG , Kristensen O , Andersen C , Alzu A , Sløk FA , Blakskjær P , Madsen D , Azevedo C , Micco I , Hansen NJV .

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DNA-encoded small molecule libraries (DELs) have facilitated the discovery of novel modulators of many different therapeutic protein targets. We report the first successful screening of a multimillion membered DEL inside a living cell. We demonstrate a novel method using oocytes from the South African clawed frog Xenopus laevis. The large size of the oocytes of 1 μL, or 100 000 times bigger than a normal somatic cell, permits simple injection of DELs, thus resolving the fundamental problem of delivering DELs across cell membranes for in vivo screening. The target protein was expressed in the oocytes fused to a prey protein, to allow specific DNA labeling and hereby discriminate between DEL members binding to the target protein and the endogenous cell proteins. The 194 million member DEL was screened against three pharmaceutically relevant protein targets, p38α, ACSS2, and DOCK5. For all three targets multiple chemical clusters were identified. For p38α, validated hits with single digit nanomolar potencies were obtained. This work demonstrates a powerful new approach to DEL screening, which eliminates the need for highly purified active target protein and which performs the screening under physiological relevant conditions and thus is poised to increase the DEL amenable target space and reduce the attrition rates.

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Species referenced: Xenopus laevis
Genes referenced: acss2.1 acss2.2 dock5 pigy

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	Graphical Abstract
	Figure 1. Cellular binder trap enrichment (cBTE) for DEL screening in a living Xenopus laevis oocyte.
	Figure 2. (A) Outline of the POI-prey constructs and their sizes. p38α and ACSS2 constructs contain the full-length proteins, whereas the DOCK5 construct contains only the Dock Homology Region 2. Prey protein is the catalytic domain of human carbonic anhydrase IX (28.1 kDa). (B) Representation of bait-DNA and the structure of bait molecule.
	Figure 3. (A) Heatmap of pairwise Tanimoto similarity coefficients (Tc): green, p38α specific hits; orange, ACSS2 specific hits; purple, DOCK5 specific hits; brown, non-POI hits. Three singleton hits were not assigned to any clusters. Annotation in blue indicates observation of hit in experiments. (B) Heatmap section with p38α hits annotated for IC50 values (green, below 10 μM; orange, 10–200 μM; gray, >200 μM) and chemical structures (asterisk indicates original DNA-attachment point).
	Figure 4. (A) Venn diagram of number of hits observed for p38α, ACSS2, and DOCK5. (B) Plot of observations numbers of hits in experiment 1 vs experiment 2. p38α specific hits are in green, and non-POI hits are in brown.