Novel PTP1B inhibitors identified by DNA display of fragment pairs.

DNA display of PNA-encoded libraries was used to pair fragments containing different phosphotyrosine surrogates with diverse triazoles. Microarray-based screening of the combinatorially paired fragment sets (62,500 combinations) against a prototypical phosphatase, PTP1B, was used to identify the fittest fragments. A focused library (10,000 members) covalently pairing identified fragments with linkers of different length and geometry was synthesized. Screening of the focused library against PTP1B and closely related TCPTP revealed orthogonal inhibitors. The selectivity of the identified inhibitors for PTP1B versus TCPT was confirmed by enzymatic inhibition assay.

Assembly of PNA-tagged small molecules, peptides, and carbohydrates onto DNA templates: programming the combinatorial pairing and inter-ligand distance.

The biochemical stability and desirable hybridization properties of peptide nucleic acids (PNA) coupled to the robustness of the peptidic chemistry involved in their oligomerization make them an attractive nucleic acid tag to encode molecules and program their assembly into higher order oligomers. The ability to program the dimerization of ligands with controlled distance between the ligands has important applications in emulating multimeric interactions. Additionally, the ability to program different permutations of ligand assemblies in a combinatorial fashion provides access to a broad diversity and offers a rapid screening method for fragment based approaches to drug discovery. Herein, we describe protocols to covalently link diverse carbohydrates, peptides, or small molecules to PNA and combinatorially assemble them in solution onto libraries of DNA templates or onto DNA microarrays using a commercial platform without recourse to specialized equipment or heavy upfront investment.

Self-assembled antibody multimers through peptide nucleic acid conjugation.

With the recent clinical success of bispecific antibodies, a strategy to rapidly synthesize and evaluate bispecific or higher order multispecific molecules could facilitate the discovery of new therapeutic agents. Here, we show that unnatural amino acids (UAAs) with orthogonal chemical reactivity can be used to generate site-specific antibody-oligonucleotide conjugates. These constructs can then be self-assembled into multimeric complexes with defined composition, valency, and geometry. With this approach, we generated potent bispecific antibodies that recruit cytotoxic T lymphocytes to Her2 and CD20 positive cancer cells, as well as multimeric antibody fragments with enhanced activity. This strategy should accelerate the synthesis and in vitro characterization of antibody constructs with unique specificities and molecular architectures.

Expanding the scope of PNA-encoded synthesis (PES): Mtt-protected PNA fully orthogonal to fmoc chemistry and a broad array of robust diversity-generating reactions.

Nucleic acid-encoded libraries are emerging as an attractive and highly miniaturized format for the rapid identification of protein ligands. An important criterion in the synthesis of nucleic acid encoded libraries is the scope of reactions that can be used to introduce molecular diversity and devise divergent pathways for diversity-oriented synthesis (DOS). To date, the protecting group strategies that have been used in peptide nucleic acid (PNA) encoded synthesis (PES) have limited the choice of reactions used in the library synthesis to just a few prototypes. Herein, we describe the preparation of PNA monomers with a protecting group combination (Mtt/Boc) that is orthogonal to Fmoc-based synthesis and compatible with a large palette of reactions that have been productively used in DOS (palladium cross-couplings, metathesis, reductive amination, amidation, heterocycle formation, nucleophilic addition, conjugate additions, Pictet-Spengler cyclization). We incorporate γ-modifications in the PNA backbone that are known to enhance hybridization and solubility. We demonstrate the robustness of this strategy with a library synthesis that is characterized by MALDI MS analysis at every step.

Discovery of an entropically-driven small molecule streptavidin binder from nucleic acid-encoded libraries.

Dehydrocholic acid was identified as a selective streptavidin binder from a PNA-tagged library. Isothermal calorimetry titration measurements showed this interaction to be entropically driven. Peptides tagged with dehydrocholic acid can be captured on a streptavidin resin and released under thermal conditions.

Selection of a synthetic glycan oligomer from a library of DNA-templated fragments against DC-SIGN and inhibition of HIV gp120 binding to dendritic cells.

We report the synthesis of a nucleic acid-encoded carbohydrate library, its combinatorial self-assembly into 37,485 pairs and a screen against DC-SIGN leading to the identification of consensus ligand motifs. A prototypical example from the selected pairs was shown to have enhanced binding. A dendrimer incorporating the selected motifs inhibited gp120's binding to dendritic cells with higher efficiency than mannan.