In Vitro Selection of a DNA-Templated Small-Molecule Library Reveals a Class of Macrocyclic Kinase Inhibitors

Abstract

DNA-templated organic synthesis enables the translation of DNA sequences into synthetic small-molecule libraries suitable for in vitro selection. Previously, we described the DNA-templated multistep synthesis of a 13 824-membered small-molecule macrocycle library. Here, we report the discovery of small molecules that modulate the activity of kinase enzymes through the in vitro selection of this DNA-templated small-molecule macrocycle library against 36 biomedically relevant protein targets. DNA encoding selection survivors was amplified by PCR and identified by ultra-high-throughput DNA sequencing. Macrocycles corresponding to DNA sequences enriched upon selection against several protein kinases were synthesized on a multimilligram scale. In vitro assays revealed that these macrocycles inhibit (or activate) the kinases against which they were selected with IC50 values as low as 680 nM. We characterized in depth a family of macrocycles enriched upon selection against Src kinase, and showed that inhibition was highly dependent on the identity of macrocycle building blocks as well as on backbone conformation. Two macrocycles in this family exhibited unusually strong Src inhibition selectivity even among kinases closely related to Src. One macrocycle was found to activate, rather than inhibit, its target kinase, VEGFR2. Taken together, these results establish the use of DNA-templated synthesis and in vitro selection to discover small molecules that modulate enzyme activities, and also reveal a new scaffold for selective ATP-competitive kinase inhibition.

Macrocyclization Reactions: The Importance of Conformational, Configurational, and Template-Induced Preorganization

“…The preparation of a DNA-templated small molecule library (41), followed by their in vitro selection using 36 protein targets, has been described in order to obtain a new class of macrocyclic kinase inhibitors 42 and 43 ( Figure 13). 92 Although the head-to-tail cyclization of the corresponding open-chain precursor is a very common approach, macrocyclizations can also be achieved by means of multiple multicomponent processes. This methodology allows preparing macrocyclic structures of different complexities from several relatively simple building blocks in a one-pot reaction.…”

Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

“…In the past few years, application of the DEL screening technology for soluble proteins has produced inhibitors against cancer and immune disorders and against therapeutic targets. These are protein kinases such as Src, MK2, Akt3, Pim1, Aurora A kinase, p38 mitogen-activated protein (MAP) kinase, and antiapoptotic protein Bcl-xL (14,15). Expanding such technology to GPCRs has the potential to yield both orthosteric and allosteric ligands.…”

Facilitating aptamer selection and collection by capillary transient isotachophoresis with laser-induced fluorescence detection

“…Over the past several decades, screening for these new molecules has spanned collections of natural products [1,2], libraries of synthetic oligonucleotides (aptamers) [3][4][5], and chemical libraries [6,7], each with its own advantages and disadvantages. Genetic screening methods are limited to nucleotides and peptides, but allow for amplification of lead molecules using PCR, while chemical screening methods apply to a wider range of molecules, but resulting leads cannot be enriched in an automated fashion [6].…”