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. 2015 Jan 1;6(1):739-744.
doi: 10.1039/c4sc01654h. Epub 2014 Sep 22.

DNA display of fragment pairs as a tool for the discovery of novel biologically active small molecules

J-P Daguer  1 , C Zambaldo  1 , M Ciobanu  2 , P Morieux  2 , S Barluenga  1 , N Winssinger  1   2
Affiliations

DNA display of fragment pairs as a tool for the discovery of novel biologically active small molecules

J-P Daguer et al. Chem Sci. .

Abstract

Fragment-based lead discovery has proven to be a powerful method in the drug discovery process. The combinatorial output that is accessible by combining fragments is very attractive; however, identifying fragment pairs that bind synergistically and linking them productively can be challenging. Several technologies have now been established to prepare and screen nucleic acid-encoded libraries (ssDNA, dsDNA, PNA), and it has been shown that pairs of molecules combined by hybridization can bind synergistically to a target. Herein we apply this concept to combinatorially pair two libraries of small molecule fragments, use the fittest fragments supplemented with closely related analogs to build a focused library covalently linking the fragments with different spacers, and apply this strategy to the discovery of a potent ligand for Hsp70.

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Figures

Fig. 1
Fig. 1. General protocol.
Fig. 2
Fig. 2. Top: microarray-based screen of combinatorially paired fragments against Hsp70-GST (125 fragment A on the horizontal lines × 500 fragment B on the vertical lines: 62 500 combinations). Bottom: structure of the fragments highlighted on top.
Scheme 1
Scheme 1. Synthesis of a 10 000-member focused library for Hsp70 (20 A fragments × 5 spacers × 100 B fragments).
Fig. 3
Fig. 3. Top: microarray-based screen of the focused library under three different conditions: Hsp70-GST, Hsp70-GST + Hsp40, and Hsp70-GST + ATP (blue circles denote the 15 highest intensity compounds). Bottom: structure of the best ligands (blue circles).
Fig. 4
Fig. 4. A. Evaluation of the ATPase inhibitor effect of compounds 1b–15b at 200 mM on Hsp70/Hsp40 (1 μM); B. same as in A for compounds 1b and 2b in dose response (0.02–200 molar equivalence).
Fig. 5
Fig. 5. Affinity pull-down of Hsp70 vs. related (Hsp90) or unrelated (carbonic anhydrase) proteins using streptavidin resin loaded with compound 1a or 2a. A. SDS PAGE (Coomassie affinity Brilliant Blue staining) of supernatant (SN) wash and eluent (E) fractions (biotin was used as a negative control); B. same experiment as in A but with crude cell lysates from HEK and Western blotting using specific monoclonal anti-Hsp70 antibody (carbonic anhydrase was used as a negative control; see Table S1 for MS-MS analysis of the eluent of 2a).
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