Design and Construction of a Focused DNA-Encoded Library for Multivalent Chromatin Reader Proteins

Abstract

Chromatin structure and function, and consequently cellular phenotype, is regulated in part by a network of chromatin-modifying enzymes that place post-translational modifications (PTMs) on histone tails. These marks serve as recruitment sites for other chromatin regulatory complexes that ‘read’ these PTMs. High-quality chemical probes that can block reader functions of proteins involved in chromatin regulation are important tools to improve our understanding of pathways involved in chromatin dynamics. Insight into the intricate system of chromatin PTMs and their context within the epigenome is also therapeutically important as misregulation of this complex system is implicated in numerous human diseases. Using computational methods, along with structure-based knowledge, we have designed and constructed a focused DNA-Encoded Library (DEL) containing approximately 60,000 compounds targeting bi-valent methyl-lysine (Kme) reader domains. Additionally, we have constructed DNA-barcoded control compounds to allow optimization of selection conditions using a model Kme reader domain. We anticipate that this target-class focused approach will serve as a new method for rapid discovery of inhibitors for multivalent chromatin reader domains.

On‐DNA Transfer Hydrogenolysis and Hydrogenation for the Synthesis of DNA‐Encoded Chemical Libraries

“…The resulting libraries can be screened for binding to protein targets by affinity selection and active compounds identified by PCR amplification and DNA sequencing. The use of DNA tags removes the need for complex sample storage and processing facilities associated with traditional compound libraries and provides one of the most efficient means of screening synthetic compounds for biological activity [5–10] …”

Highly efficient on-DNA amide couplings promoted by micelle forming surfactants for the synthesis of DNA encoded libraries

“…These libraries can be screened against a protein target by affinity selection followed by PCR amplication and DNA sequencing to identify binders, which are then synthesised "off-DNA". [5][6][7][8][9][10] A signicant limitation of the approach currently is the range and efficiency of the chemical reactions that can be carried out on DNA-tagged molecules, i.e. reaction media (traditionally aqueous) must be compatible with DNA and must not employ reagents that react with DNA.…”

“…19 Moreover, the coupling is almost always reported with the amine component attached to the DNA (representing synthesis in the "C-to-N" direction for peptide couplings) with the acid monomers employed in large excess. 10,[20][21][22][23][24] Reports of couplings of DNA-conjugated carboxylates to amines building blocks (N-to-C) are severely limited 25 and, in our hands, we have found extension to drug-like amine building blocks to be very challenging. A recent study showed that for a range of amines, on-DNA N-to-C amide couplings only gave >70% conversion in 30% of cases.…”

On‐DNA Transfer Hydrogenolysis and Hydrogenation for the Synthesis of DNA‐Encoded Chemical Libraries

“…The use of DNA tags removes the need for complex sample storage and processing facilities associated with traditional compound libraries and provides one of the most efficient means of screening synthetic compounds for biological activity. [5][6][7][8][9][10] The success of the approach depends critically on the efficiency of the chemistry that is used to construct the DEL. "On-DNA" chemistry is usually carried out in water, due to the insolubility of DNA-conjugates in organic solvents, and the presence of DNA obviates the use of many commonly employed reagents, including acids, oxidising agents and strong bases.…”

“…The use of DNA tags removes the need for complex sample storage and processing facilities associated with traditional compound libraries and provides one of the most efficient means of screening synthetic compounds for biological activity. [ 5 , 6 , 7 , 8 , 9 , 10 ]…”