Hit‐Validation Methodologies for Ligands Isolated from DNA‐Encoded Chemical Libraries

Abstract

DNA-encoded chemical libraries (DECLs) are large collections of compounds linked to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of interest. In typical DECL selections, preferential binders are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified in this procedure need to be confirmed, by resynthesis and by performing affinity measurements. In this article we present new methods based on hybridization of oligonucleotide conjugates with fluorescently labeled complementary oligonucleotides; these facilitate the determination of affinity constants and kinetic dissociation constants. The experimental procedures were demonstrated with acetazolamide, a binder to carbonic anhydrase IX with a dissociation constant in the nanomolar range. The detection of binding events was compatible not only with fluorescence polarization methodologies, but also with Alphascreen technology and with microscale thermophoresis.

DNA‐encoded chemical libraries – achievements and remaining challenges

“…At present, the optimization of linkers connecting synergistic building blocks is still empirical, even though "on-DNA" procedures may facilitate this experimental task [22,25]. It remains to be seen whether linker optimization can be embedded in library construction and selection procedures.…”

“…In general, it is good practice to confirm the enrichment results by resynthesis of the hit compounds and by dedicated affinity measurements ("hit validation"). In addition to compound resynthesis, hit validation strategies performed "on DNA" can be considered, as the nucleic acid may facilitate resynthesis, MS detection, solubility and affinity measurements with certain experimental procedures [24,25]. The scope of DNA-encoded chemical library technology has been extended by exploiting the chemical opportunities offered by peptide nucleic acids (PNAs) [26].…”

Impact of a Central Scaffold on the Binding Affinity of Fragment Pairs Isolated from DNA‐Encoded Self‐Assembling Chemical Libraries

“…Figure 1s hows the fingerprinto ft he ESAC library selection against AGP.S creening of al ibrary,c onsisting of 550 diversity elements Ap aired to 428 diversity elements B, allowed the identification of pair A 117 /B 217 ,w hich was preferentially enriched relative to all other library members. [51] The best dissociation constant (K D = 9.9 nm)w as observed for Scaffold 1( S1), while the worst dissociation constant (K D = 1306 nm)w as found for Scaffold 11 (S11). To convert the enriched binding pair into discrete organic molecules, featuring ad irect linkage of the A 117 and B 217 moieties, as tepwise procedure was developed.…”

“…Our results show that the scaffolding moiety,u sed for fragment linking,h as as ubstantial impact on the binding affinity to the protein target. [51] The best ligands could then be resynthesized "off-DNA", yielding organic molecules devoid of any nucleic acid component. [22][23][24]50] Large combinatorial libraries can be produced by the self-assembly of partially complementary oligonucleotide conjugates.…”

“…[51] The best ligands could then be resynthesized "off-DNA", yielding organic molecules devoid of any nucleic acid component. The oligonucleotide conjugates were subsequently paired to af luorescently labeled complementary lockedn ucleic acid (LNA).…”