Synthetic Ligands Discovered by in Vitro Selection

Abstract

The recognition and catalytic properties of biopolymers derive from an elegant evolutionary mechanism, whereby the genetic material encoding molecules with superior functional attributes survives a selective pressure and is propagated to subsequent generations. This process is routinely mimicked in vitro to generate nucleic-acid or peptide ligands and catalysts. Recent advances in DNAprogrammed organic synthesis have raised the possibility that evolutionary strategies could also be used for small-molecule discovery, but the idea remains unproven. Here, using DNA-programmed combinatorial chemistry, a collection of 100 million distinct compounds is synthesized and subjected to selection for binding to the N-terminal SH3 domain of the proto-oncogene Crk. Over six generations, the molecular population converges to a small number of novel SH3 domain ligands. Remarkably, the hits bind with affinities similar to those of peptide SH3 ligands isolated from phage libraries of comparable complexity. The evolutionary approach has the potential to drastically simplify and accelerate small-molecule discovery.

Synthesis at the Interface of Chemistry and Biology

“…A similar approach has been applied to natural mutations in nuclear receptors or enzymes 303 and can likely be applied to other enzymes and receptors, such as lipid kinases and methyltransferases. A number of laboratories including those of Liu and Harbury have created libraries of small molecules by templated organic synthesis in which a DNA template is used to direct the stepwise synthesis of molecules using duplex formation to create high effective molarities of reactants 28,29,304,305 (Figure 18). This approach is being used to search for selective ligands to biological receptors, as well as identify novel chemical reactions, and may one day lead to synthetic libraries which can be encoded, amplified, chemically mutated and selected in a process that mimics natural protein evolution.…”

Novel selection methods for DNA-encoded chemical libraries

“…A target protein is immobilized by binding or covalent attachment to a solid support and then incubated with a DNA-encoded chemical library. Alternatively, the library can be incubated with free protein in solution and captured with target-specific antibodies that are immobilized or tagged with an affinity handle [1,16]. After washing [7], library members capable of binding the target are eluted and subjected to additional round(s) of selection or PCR amplification of their associated DNA templates and massively parallel high-throughput DNA sequencing.…”

Chemistry on Nucleic Acid Templates

“…After converting the singlestranded DNA to a duplex, only two consecutive rounds of section for 3-E7 antibody binding were sufficient to converge the population, with > 70% of the sequences encoding [Leu]enkephalin or single mutants [118]. In a recent follow-up report, Wrenn, Harbury, and co-workers synthesized a far more complex library containing 10 8 DNAlinked octamer peptoids (N-alkylated peptides), which were coupled in a DNA-routed two-step procedure, involving a-chloracetylation of the terminal amine, followed by nucleophilic substitution with the next amine building block [127]. Coupling yields over both steps averaged at 85 -90%.…”

“…DNA Hybridization is not relevant during bond-formation, making a vast chemical repertoire available for the synthesis of encoded libraries [11]. DNA-Displayed libraries have been constructed using peptide-coupling reactions, nucleophilic displacement reactions involving amines and thiols, reductive aminations, electrophilic aromatic substitutions, periodate oxidations, and phosphine-based reductions [11] [125] [127], without detectable sign of DNA or peptide side-chain modification, as evident from MS, Edman degradation, and binding characteristics to the 3-E7 antibody [124].…”

“…Larger libraries are currently reserved to biochemical display technologies (10 13 ) [123] or NAs (10 15 ) [88]. With no restrictions on the template architecture, readily available reagents that do not require DNA conjugation, and compatibility with genetic technologies like DNA shuffling [129], DNA-display libraries with up to 10 13 members could be constructed and selected for drug-like small molecules [130] with K D values in the nm range [11] [127]. DNA Display plays to its strengths where DTS shows its major drawbacks; likewise, DTS products contain more drug-like structures, proximity effects enable to control reactivity in ways that are not accessible in a traditional synthesis, and do not require specialized equipment for liquid handling like DNA display, making the two promising technologies complementary approaches for the synthesis and selection of DNAencoded libraries of small molecules.…”