Hydrogel-Encapsulated Beads Enable Proximity-Driven Encoded Library Synthesis and Screening.

Encoded combinatorial library technologies have dramatically expanded the chemical space for screening but are usually only analyzed by affinity selection binding. It would be highly advantageous to reformat selection outputs to "one-bead-one-compound" solid-phase libraries, unlocking activity-based and cellular screening capabilities. Here, we describe hydrogel-encapsulated magnetic beads that enable such a transformation. Bulk emulsion polymerization of polyacrylamide hydrogel shells around magnetic microbeads yielded uniform particles (7 ± 2 µm diameter) that are compatible with diverse in-gel functionalization (amine, alkyne, oligonucleotides) and transformations associated with DNA-encoded library synthesis (acylation, enzymatic DNA ligation). In a case study of reformatting mRNA display libraries, transcription from DNA-templated magnetic beads encapsulated in gel particles colocalized both RNA synthesis via hybridization with copolymerized complementary DNA and translation via puromycin labeling. Two control epitope templates (V5, HA) were successfully enriched (50- and 99-fold, respectively) from an NNK5 library bead screen via FACS. Proximity-driven library synthesis in concert with magnetic sample manipulation provides a plausible means for reformatting encoded combinatorial libraries at scale.

Liposomal Permeation Assay for Droplet-Scale Pharmacokinetic Screening.

Combinatorial library screening increasingly explores chemical space beyond the Ro5 (bRo5), which is useful for investigating "undruggable" targets but suffers compromised cellular permeability and therefore bioavailability. Moreover, structure-permeation relationships for bRo5 molecules are unclear partially because high-throughput permeation measurement technology for encoded combinatorial libraries is still nascent. Here, we present a permeation assay that is scalable to combinatorial library screening. A liposomal fluorogenic azide probe transduces permeation of alkyne-labeled molecules into small unilamellar vesicles via copper-catalyzed azide-alkyne cycloaddition. Control alkynes (e.g., propargylamine, various alkyne-labeled PEGs) benchmarked the assay. Cell-permeable macrocyclic peptides, exemplary bRo5 molecules, were alkyne labeled and shown to retain permeability. The assay was miniaturized to microfluidic droplets with high assay quality (Z' ≥ 0.5), demonstrating excellent discrimination of photocleaved known membrane-permeable and -impermeable model library beads. Droplet-scale permeation screening will enable pharmacokinetic mapping of bRo5 libraries to build predictive models.

Evaluating Translational Efficiency of Noncanonical Amino Acids to Inform the Design of Druglike Peptide Libraries.

Advances in genetic code reprogramming have allowed the site-specific incorporation of noncanonical functionalities into polypeptides and proteins, providing access to wide swaths of chemical space via in vitro translation techniques like mRNA display. Prior efforts have established that the translation machinery can tolerate amino acids with modifications to both the peptide backbone and side chains, greatly broadening the chemical space that can be interrogated in ligand discovery efforts. However, existing methods for confirming the translation yield of new amino acid building blocks for these technologies necessitate multistep workups and, more importantly, are not relevant for measuring translation within the context of a combinatorial library consisting of multiple noncanonical amino acids. In this study, we developed a luminescence-based assay to rapidly assess the relative translation yield of any noncanonical amino acid in real time. Among the 59 amino acids tested here, we found that many translate with high efficiency, but translational yield is not necessarily correlated to whether the amino acid is proteinogenic or has high tRNA acylation efficiency. Interestingly, we found that single-template translation data can inform the library-scale translation yield and that shorter peptide libraries are more tolerant of lower-efficiency amino acid monomers. Together our data show that the luminescence-based assay described herein is an essential tool in evaluating new building blocks and codon table designs within mRNA display toward the goal of developing druglike peptide-based libraries for drug discovery campaigns.

Discovery and molecular basis of subtype-selective cyclophilin inhibitors.

Although cyclophilins are attractive targets for probing biology and therapeutic intervention, no subtype-selective cyclophilin inhibitors have been described. We discovered novel cyclophilin inhibitors from the in vitro selection of a DNA-templated library of 256,000 drug-like macrocycles for cyclophilin D (CypD) affinity. Iterated macrocycle engineering guided by ten X-ray co-crystal structures yielded potent and selective inhibitors (half maximal inhibitory concentration (IC50) = 10 nM) that bind the active site of CypD and also make novel interactions with non-conserved residues in the S2 pocket, an adjacent exo-site. The resulting macrocycles inhibit CypD activity with 21- to >10,000-fold selectivity over other cyclophilins and inhibit mitochondrial permeability transition pore opening in isolated mitochondria. We further exploited S2 pocket interactions to develop the first cyclophilin E (CypE)-selective inhibitor, which forms a reversible covalent bond with a CypE S2 pocket lysine, and exhibits 30- to >4,000-fold selectivity over other cyclophilins. These findings reveal a strategy to generate isoform-selective small-molecule cyclophilin modulators, advancing their suitability as targets for biological investigation and therapeutic development.

Multimodal small-molecule screening for human prion protein binders.

Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including 19F-observed and saturation transfer difference (STD) NMR spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and in silico screening. A single benzimidazole compound was confirmed in concentration-response, but affinity was very weak (Kd > 1 mm), and it could not be advanced further. The exceptionally low hit rate observed here suggests that PrP is a difficult target for small-molecule binders. Whereas orthogonal binder discovery methods could yield high-affinity compounds, non-small-molecule modalities may offer independent paths forward against prion disease.

Author Correction: Second-generation DNA-templated macrocycle libraries for the discovery of bioactive small molecules.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Second-generation DNA-templated macrocycle libraries for the discovery of bioactive small molecules.

DNA-encoded libraries have emerged as a widely used resource for the discovery of bioactive small molecules, and offer substantial advantages compared with conventional small-molecule libraries. Here, we have developed and streamlined multiple fundamental aspects of DNA-encoded and DNA-templated library synthesis methodology, including computational identification and experimental validation of a 20 × 20 × 20 × 80 set of orthogonal codons, chemical and computational tools for enhancing the structural diversity and drug-likeness of library members, a highly efficient polymerase-mediated template library assembly strategy, and library isolation and purification methods. We have integrated these improved methods to produce a second-generation DNA-templated library of 256,000 small-molecule macrocycles with improved drug-like physical properties. In vitro selection of this library for insulin-degrading enzyme affinity resulted in novel insulin-degrading enzyme inhibitors, including one of unusual potency and novel macrocycle stereochemistry (IC50 = 40 nM). Collectively, these developments enable DNA-templated small-molecule libraries to serve as more powerful, accessible, streamlined and cost-effective tools for bioactive small-molecule discovery.

Discovery of a Covalent Kinase Inhibitor from a DNA-Encoded Small-Molecule Library × Protein Library Selection.

We previously reported interaction determination using unpurified proteins (IDUP), a method to selectively amplify DNA sequences encoding ligand:target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. In this study, we applied IDUP to libraries of DNA-encoded bioactive compounds and DNA-tagged human kinases to identify ligand:protein binding partners out of 32 096 possible combinations in a single solution-phase library × library experiment. The results recapitulated known small molecule:protein interactions and also revealed that ethacrynic acid is a novel ligand and inhibitor of MAP2K6 kinase. Ethacrynic acid inhibits MAP2K6 in part through alkylation of a nonconserved cysteine residue. This work validates the ability of IDUP to discover ligands for proteins of biomedical relevance.

Structural and Biochemical Basis for Intracellular Kinase Inhibition by Src-specific Peptidic Macrocycles.

Protein kinases are attractive therapeutic targets because their dysregulation underlies many diseases, including cancer. The high conservation of the kinase domain and the evolution of drug resistance, however, pose major challenges to the development of specific kinase inhibitors. We recently discovered selective Src kinase inhibitors from a DNA-templated macrocycle library. Here, we reveal the structural basis for how these inhibitors retain activity against a disease-relevant, drug-resistant kinase mutant, while maintaining Src specificity. We find that these macrocycles display a degree of modularity: two of their three variable groups interact with sites on the kinase that confer selectivity, while the third group interacts with the universally conserved catalytic lysine and thereby retains the ability to inhibit the "gatekeeper" kinase mutant. We also show that these macrocycles inhibit migration of MDA-MB-231 breast tumor cells. Our findings establish intracellular kinase inhibition by peptidic macrocycles, and inform the development of potent and specific kinase inhibitors.

Novel selection methods for DNA-encoded chemical libraries.

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries.